Methods for treatment of oncological disorders using epimetabolic shifters, multidimensional intracellular molecules, or environmental influencers

ABSTRACT

Methods and formulations for treating oncological disorders in humans using epimetabolic shifters, multidimensional intracellular molecules or environmental influencers are described.

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application Ser.No. 61/177,241, filed May 11, 2009, entitled “Methods for Treatment ofOncological Disorders Using an Epimetabolic Shifter (Coenzyme Q10)”(Attorney Docket No.: 117732-00601), U.S. Provisional Application Ser.No. 61/177,243, filed May 11, 2009, entitled “Methods for Treatment ofOncological Disorders Using Epimetabolic Shifters, MultidimensionalIntracellular Molecules or Environmental Influencers” (Attorney DocketNo.: 117732-00701), U.S. Provisional Application Ser. No. 61/177,244,filed May 11, 2009, entitled “Methods for the Diagnosis of OncologicalDisorders Using Epimetabolic Shifters, Multidimensional IntracellularMolecules or Environmental Influencers” (Attorney Docket No.:117732-00801), U.S. Provisional Application Ser. No. 61/177,245, filedMay 11, 2009, entitled “Methods for Treatment of Metabolic DisordersUsing Epimetabolic Shifters, Multidimensional Intracellular Molecules orEnvironmental Influencers” (Attorney Docket No.: 117732-00901), and U.S.Provisional Application Ser. No. 61/177,246, filed May 11, 2009,entitled “Methods for the Diagnosis of Metabolic Disorders UsingEpimetabolic Shifters, Multidimensional Intracellular Molecules orEnvironmental Influencers” (Attorney Docket No.: 117732-01001). Theentire contents of each of the foregoing applications are herebyincorporated herein by reference.

BACKGROUND OF THE INVENTION

Cancer is presently one of the leading causes of death in developednations and is a serious threat to modern society. Cancer can develop inany tissue of any organ at any age. Worldwide, more than 10 millionpeople are diagnosed with cancer every year and it is estimated thatthis number will grow to 15 million new cases every year by 2020. It isbelieved that cancer causes six million deaths every year or 12% of thedeaths worldwide.

The etiology of cancer is not clearly understood. Cancer has been linkedto or associated with many factors over the many years of ongoingresearch including genetic susceptibility, chromosome breakagedisorders, viruses, environmental factors and immunologic disorders.Cancer encompasses a large category of medical conditions. Cancer cellscan arise in almost any organ and/or tissue of the body. Cancer developswhen cells in a part of the body begin to grow or differentiate out ofcontrol.

Although recent research has vastly increased our understanding of manyof the molecular mechanisms of tumorigenesis and has provided numerousnew avenues for the treatment of cancer, standard treatments for mostmalignancies remain gross resection, chemotherapy, and radiotherapy.While increasingly successful, each of these treatments may causenumerous undesired side effects. For example, surgery may result inpain, traumatic injury to healthy tissue, and scarring. Radiationtherapy has the advantage of killing cancer cells but it also damagesnon-cancerous tissue at the same time. Chemotherapy involves theadministration of various anti-cancer drugs to a patient. These standardtreatments often are accompanied by adverse side effects, e.g., nausea,immune suppression, gastric ulceration and secondary tumorigenesis.

Over the years, many individuals and companies have conducted extensiveresearch searching for improvements in the treatments for the wide arrayof cancers. Companies are developing bioactive agents including chemicalentities, e.g., small molecules, and biologics, e.g., antibodies, withthe desire of providing more beneficial therapies for cancer. Some ofthe bioactive agents tested have worked and provided beneficialtherapeutic effects in some individuals or cancer types and others havefailed or had minimal therapeutic effects in their testing protocols.Other bioactive agents studied to date have mechanisms of action thatare not entirely understood.

Coenzyme Q10, also referred to herein as CoQ10, Q10, ubiquinone, orubidecarenone, is a popular nutritional supplement and can be found incapsule form in nutritional stores, health food stores, pharmacies, andthe like, as a vitamin-like supplement to help protect the immune systemthrough the antioxidant properties of ubiquinol, the reduced form ofCoQ10. CoQ10 is art-recognized and further described in InternationalPublication No. WO 2005/069916, the entire disclosure of which isincorporated by reference herein.

CoQ10 is found throughout most tissues of the human body and the tissuesof other mammals. The tissue distribution and redox state of CoQ10 inhumans has been reviewed in a review article by Bhagavan H N, et al.,Coenzyme Q10: Absorption, tissue uptake, metabolism and pharmacokinetic,Free Radical Research 40(5), 445-453 (2006) (hereinafter, Bhagavan, etal.). The authors report that “as a general rule, tissues withhigh-energy requirements or metabolic activity such as the heart,kidney, liver and muscle contain relatively high concentrations ofCoQ10.” The authors further report that “[a] major portion of CoQ10 intissues is in the reduced form as the hydroquinone or uniquinol, withthe exception of brain and lungs,” which “appears to be a reflection ofincreased oxidative stress in these two tissues.” In particular,Bhagavan et al. reports that in heart, kidney, liver, muscle, intenstineand blood (plasma), about 61%, 75%, 95%, 65%, 95% and 96%, respectively,of CoQ10 is in the reduced form. Similarly, Ruiz-Jiminez, et al.,Determination of the ubiquinol-10 and ubiquinone-10 (coenzyme Q10) inhuman serum by liquid chromatography tandem mass spectrometry toevaluate the oxidative stress, J. Chroma A 1175(2), 242-248 (2007)(hereinafter Ruiz-Jiminez, et al.) reports that when human plasma wasevaluated for Q10 and the reduced form of Q10 (Q10H2), the majority(90%) of the molecule was found in the reduced form.

CoQ10 is very lipophilic and, for the most part, insoluble in water. Dueto its insolubility in water, limited solubility in lipids, andrelatively large molecular weight, the efficiency of absorption oforally administered CoQ10 is poor. Bhagavan, et al. reports that “in onestudy with rats it was reported that only about 2-3% oforally-administered CoQ10 was absorbed.” Bhagavan, et al. furtherreports that “[d]ata from rat studies indicate that CoQ10 is reduced toubiquinol either during or following absorption in the intestine.”

CoQ10 has been associated with cancer in the literature for many years.Described below are some representative but not all inclusive examplesof the reported associations in the literature. Karl Folkers, et al.,Survival of Cancer Patients on Therapy with Coenzyme Q10, Biochemicaland Biophysical Research Communication 192, 241-245 (1993) (herein after“Folkers, et al.”) describes eight case histories of cancer patients “ontherapy with CoQ10” and their stories of survival “for periods of 5-15years.” CoQ10 was orally administered to eight patients having differenttypes of cancer, including pancreatic carcinoma, adenocarcinoma,laryngeal carcinoma, breast, colon, lung and prostate cancer. Folkers,et al. sets forth that “these results now justify systemic protocols.”Lockwood, et al., Progress on Therapy of Breast Cancer with Vitamin Q10and the Regression of Metastases, Biochemical and Biophysical ResearchCommunication 212, 172-177 (1995) (hereinafter “Lockwood, et al.”) isanother review article that reports on the “[p]rogress on therapy ofbreast cancer with Vitamin Q10”. Lockwood, et al. refers to Folkers, etal., which “covers 35 years of international research on animals andhumans which revealed variable levels of vitamin Q10 in non-tumor andtumor tissues and includes data on vitamin Q10 which are intrinsic tothe host defense system as based on increased survivors of treated micewith tumors”. Lockwood, et al. further sets forth that “[t]he potentialof vitamin Q10 therapy of human cancer became evident in 1961” relyingon a study that determined the blood levels of CoQ10 in 199 Swedish andAmerican cancer patients that revealed variable levels of deficienciesin cases of breast cancer. U.S. Pat. No. 6,417,233, issued Jul. 9, 2002(hereinafter Sears, et al.) describes compositions containinglipid-soluble benzoquinones, e.g., coenzyme Q10, for the preventionand/or treatment of mitochondriopathies. Sears, et al. sets forth that“CoQ10 treatment has been reported to provide some benefits in cancerpatients (see column 2, lines 30-31).”

As of the date of filing of this application, the National CancerInstitute reports that no well-designed clinical trials involving largenumbers of patients of CoQ10 in cancer treatment have been conductedsince “the way the studies were done and the amount of informationreported made it unclear if the benefits were caused by the coenzyme Q10or by something else.” See The National Cancer Institute (NCI),available at www dot cancer dot gov slash cancertopics slash pdq slashcam salsh coenzymeQ10 slash patient slash allpages (Sep. 29, 2008). Inparticular, the NCI cites three small studies on the use of CoQ10 as anadjuvant therapy after standard treatment in breast cancer patients, inwhich some patients appeared to be helped by the treatment, andreiterates that “weaknesses in study design and reporting, however, madeit unclear if benefits were caused by the coenzyme Q10 or by somethingelse.” The NCI specifies that “these studies had the followingweaknesses: the studies were not randomized or controlled; the patientsused other supplements in addition to coenzyme Q10; the patientsreceived standard treatments before or during the coenzyme Q10 therapy;and details were not reported for all patients in the studies.” The NCIfurther reports on “anecdotal reports that coenzyme Q10 has helped somecancer patients live longer, including patients with cancers of thepancreas, lung, colon, rectum and prostate,” but states that ‘thepatients described in these reports, however, also received treatmentsother than coenzyme Q10 including chemotherapy, radiation therapy andsurgery.”

US Patent Application Publication 2006/0035981, published Feb. 16, 2006(hereinafter “Mazzio 2006”) describes methods and formulations fortreating or preventing human and animal cancers using compositions thatexploit the vulnerability of cancers with regards to its anaerobicrequirement for non-oxidative phosphorylation of glucose to deriveenergy, which is opposite to the host. The formulations of Mazzio 2006contain one or more compounds that synergistically promote oxidativemetabolism and/or impede lactic acid dehydrogenase or anaerobic glucosemetabolism and more particularly are described as containing“2,3-dimethoxy-5-methyl-1,4-benzoquinone (herein also termed “DMBQ”)(quinoid base) and options for the entire ubiquinone series includingcorresponding hydroquinones, ubichromenols, ubichromanols orsynthesized/natural derivatives and analogues. See Mazzio 2006 at page3, paragraph 0010. Mazzio 2006 establishes “the short chain ubiquinones(CoQ<3) as anti-cancer agents and even further establishes that“2,3-dimethoxy-5-methyl-1,4-benzoquinone (DMBQ) is in excess of 1000times more potent than CoQ10 as an anti-cancer agent.” See Mazzio 2006at page 3, paragraph 0011. Mazzio 2006 further set forth that the study“did not find CoQ10 to be as lethal as expected” and like “previousstudies that have employed CoQ10 against cancer have been somewhatcontradictory”. See Mazzio 2006 at pages 3-4 for an extensive list ofcitations supporting this statement.

US Patent Application Publication 2007/0248693, published Oct. 25, 2007(herein after “Mazzio 2007”) also describes nutraceutical compositionsand their use for treating or preventing cancer. Again, this publishedpatent application focuses on the short chain ubiquinones andspecifically sets forth that CoQ10 is not a critical component of thisinvention. According to Mazzio 2007 “while CoQ10 can increase the Vmaxof mitochondrial complex II activity in cancer cells (Mazzio andSoliman, Biochem Pharmacol. 67:1167-84, 2004), this did not control therate of mitochondrial respiration or O2 utilization through complex IV.And, CoQ10 was not as lethal as expected. Likewise, results of CoQ10against cancer have been contradictory.” See Mazzio 2007 at page 5,paragraph 0019.

SUMMARY OF THE INVENTION

Applicants have previously described topical formulations of CoQ10 andmethods for reducing the rate of tumor growth in animal subjects (Hsiaet al., WO 2005/069916 published Aug. 4, 2005). In the experimentsdescribed in Hsia et al., CoQ10 was shown to increase the rate ofapoptosis in a culture of skin cancer cells but not normal cells.Moreover, treatment of tumor-bearing animals with a topical formulationof CoQ10 was shown to dramatically reduce the rate of tumor growth inthe animals.

The present invention is based, at least in part, upon a more completeunderstanding of the role of CoQ10 within a human and/or cell. Inparticular, the methods and formulations of the present invention arebased, at least in part, upon the knowledge gained about the therapeuticactivity of CoQ10 for oncological disorders learned by designing andimplementing human clinical trials and/or by administering CoQ10 tohuman subjects and observing the surprising and unexpected results thatoccur during these trials and/or treatment regimens. The methods andformulations of the present invention are further based, at least inpart, upon insight gained into the therapeutic mechanism of CoQ10 fromextensive studies of CoQ10 treatment of cells in vitro.

Specifically, in at least one embodiment, the methods and formulationsof the present invention are based, at least in part, on the surprisingdiscovery that application of Coenzyme Q10 (also referred to as CoQ10 orQ10 herein) to cells results in selective induction of an apoptoticresponse in cancer cells, with no effect or, in some cases, a positiveeffect on growth of normal cells. Moreover, in at least one additionalembodiment, it was unexpectedly found that cell lines derived fromaggressive cancers were more sensitive to CoQ10 (e.g., required lowerconcentrations and/or treatment time of CoQ10 for cytotoxicity and/orinduction of apoptosis) as compared to cell lines derived from lessaggressive or non-aggressive cancers. A time and dose response ofmitochondrial Q10 levels was observed, wherein after 48 hours, the levelof Q10 in cell mitochondria was increased by six fold. In at least oneadditional embodiment, the invention is further based on the surprisingand unexpected discovery that the Q10 is maintained in the suppliedoxidized form (pro-oxidant) and not converted to the reduced(anti-oxidant) form of Q10H2 in any significant amounts. In anotherembodiment, the invention is still further based on the discovery thatthe expression of a significant number of genes are modulated in cellstreated with the oxidized from of Q10. These modulated proteins werefound to be clustered into several cellular pathways, includingapoptosis, cancer biology and cell growth, glycolysis and metabolism,molecular transport, and cellular signaling.

Taken together, the results described herein have provided insight intothe therapeutic mechanism of Q10. For example, while not wishing to bebound by theory, Applicants' discoveries indicate that Q10 and, inparticular, the oxidized form of Q10, induces a metabolic shift to thecell microenvironment. Differential metabolism is known to occur incancer cells (the Warburg effect), whereby most cancer cellspredominantly produce energy by glycolysis followed by lactic acidfermentation in the cytosol, rather than by oxidative phosphorylation(oxidation of pyruvate) in the mitochondria. Applicants' discoveriesindicate that Q10 is capable of shifting the metabolic state of cancercells from anaerobic use of glucose to mitochondrial oxidativephosphorylation.

Based on Applicants' data presented herein, Q10 has been identified as aMultidimensional Intracellular Molecule (MIM) and as an EpimetabolicShifter (Epi-Shifter). The present invention provides methods foridentifying other MIMs and/or Epi-shifters. The present inventionfurther provides MIMs, Epi-shifters and methods for treating anoncological disorder by using same.

Accordingly, the invention provides, in a first aspect, a method fortreating or preventing an oncological disorder in a human, the methodcomprising administering an environmental influencer (env-influencer) tothe human in an amount sufficient to treat or prevent the oncologicaldisorder, wherein the env-influencer is not Coenzyme Q10, therebytreating or preventing the oncological disorder in the human.

In a related aspect, the invention provides a method for treating,alleviating symptoms of, inhibiting progression of, or preventing anoncological disorder in a mammal, the method comprising: administeringto the mammal in need thereof a therapeutically effective amount ofpharmaceutical composition comprising at least one environmentalinfluencer (env-influencer), wherein the environmental influencerselectively elicits, in a cancerous cell of the mammal, a cellularmetabolic energy shift from glycolysis to mitochondrial oxidativephosphorylation, towards levels observed in a normal cell of the mammalunder normal physiological conditions.

As used herein, “glycolysis” optionally includes the associated lactatebiosynthesis that produces lactate from pyruvate.

In certain embodiments, the environmental influencer does notsubstantially elicit, in non-cancerous cells of the mammal, the cellularmetabolic energy shift from glycolysis to mitochondrial oxidativephosphorylation.

In certain embodiments, the mammal is human, or a non-human mammal.

In certain embodiments, the environmental influencer is not CoenzymeQ10, or its metabolites or analogs thereof (including analogs having noor at least one isoprenyl repeats).

In certain embodiments, the oncological disorder is responsive orsensitive to treatment by Coenzyme Q10 or its metabolites or analogsthereof.

In certain embodiments, the environmental influencer induces apoptosisor cell death mechanism in the cancerous cell.

In certain embodiments, the environmental influencer inhibitsangiogenesis in the cancerous cell.

In certain embodiments, the environmental influencer induces amodulation of the immune-related elements within the microenvironment inthe cancerous cell.

In certain embodiments, the environmental influencer induces a change incell cycle control in the cancerous cell.

In certain embodiments, the environmental influencer comprises: (a)benzoquinone or at least one molecule that facilitates the biosynthesisof the benzoquinone ring, and (b) at least one molecule that facilitatesthe synthesis of and/or attachment of isoprenoid units to thebenzoquinone ring.

In certain embodiments, the at least one molecule that facilitates thebiosynthesis of the benzoquinone ring comprises: L-Phenylalanine,DL-Phenylalanine, D-Phenylalanine, L-Tyrosine, DL-Tyrosine, D-Tyrosine,4-hydroxy-phenylpyruvate, 3-methoxy-4-hydroxymandelate(vanillylmandelate or VMA), vanillic acid, pyridoxine, or panthenol.

In certain embodiments, the at least one molecule that facilitates thesynthesis of and/or attachment of isoprenoid units to the benzoquinonering comprises: phenylacetate, 4-hydroxy-benzoate, mevalonic acid,acetylglycine, acetyl-CoA, or farnesyl.

In certain embodiments, the environmental influencer comprises: (a) oneor more of L-Phenylalanine, L-Tyrosine, and 4-hydroxyphenylpyruvate;and, (b) one or more of 4-hydroxy benzoate, phenylacetate, andbenzoquinone.

In certain embodiments, the environmental influencer: (a) inhibits Bcl-2expression and/or promotes Caspase-3 expression; and/or, (b) inhibitscell proliferation.

In one embodiment, the env-influencer is a multidimensionalintracellular molecule (MIM). In certain embodiments, the MIM isselected from: alpha ketoglutarate/alpha ketoglutaric acid, Malate/Malicacid, Succinate/Succinic acid, Glucosamine, Adenosine, AdenosineDiphosphate, Glucuronide/Glucuronic acid, Nicotinic Acid, Nicotinic AcidDinucleotide, Alanine/Phenylalanine, Pyridoxine, Thiamine, or FlavinAdenine Dinucleotide. In one embodiment, the MIM is selected from thegroup consisting of acetyl Co-A, palmityl Co-A, L-carnitine, and aminoacids (e.g., tyrosine, phenylalanine, and cysteine).

In one embodiment, the env-influencer is an epimetabolic shifter(epi-shifter). In certain embodiments, the epimetabolic shifter isselected from: Transaldolase, Transketolase, Succinyl CoA synthase,Pyruvate Carboxylase, or Riboflavin. In one embodiment, the epishifteris selected from the group consisting of vitamin D3 and ECM components.In one embodiment, the ECM components are selected from the groupconsisting of fibronectin; immunomodulators (e.g., TNFα or any of theinterleukins, e.g., IL-5, IL-12, IL-23) angiogenic factors; andapoptotic factors.

In one embodiment, a population of humans are treated and at least 25%of the population had a systemic environmental influencer (e.g.,Coenzyme Q10) level that was therapeutic for the disorder being treated.In other embodiments, a population of humans are treated and at least5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95% or more of the population had a systemicenvironmental influencer (e.g., Coenzyme Q10) level that was therapeuticfor the disorder being treated. It should be understood that rangeshaving any one of these values as the upper or lower limits are alsointended to be part of this invention, e.g., 10% to 25%, 15% to 35%, 25%to 50%, 35% to 60%, 40% to 70%, 50% to 75%, 60% to 85% or 70% to 90%.

In one embodiment, a population of humans are treated and at least 25%of the population had a diminishment of symptoms as measured byart-recognized endpoints including tissue pathology, clinicalobservations, photographic analyses, CT-scan, MRI imaging, blood, serumor plasma markers of cancer.

In one embodiment, a population of humans are treated and at least 50%of the population had a diminishment of symptoms as measured byart-recognized endpoints including tissue pathology, clinicalobservations, photographic analyses, CT-scan, MRI imaging, blood, serumor plasma markers of cancer.

In other embodiments, a population of humans are treated and at least5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, 98% or more of the population had adiminishment of symptoms as measured by art-recognized endpointsincluding tissue pathology, clinical observations, photographicanalyses, CT-scan, MRI imaging, blood, serum or plasma markers ofcancer. It should be understood that ranges having any one of thesevalues as the upper or lower limits are also intended to be part of thisinvention, e.g., 10% to 25%, 15% to 35%, 25% to 50%, 35% to 60%, 40% to70%, 50% to 75%, 60% to 85% or 70% to 90%.

In various embodiments, the population of humans treated may be about 3patients, about 5 patients, about 10 patients, about 15 patients, about20 patients, about 25 patients, about 30 patients, about 35 patients,about 40 patients, about 50 patients, about 60 patients, about 70patients, about 80 patients, about 90 patients, about 100 patients,about 125 patients, about 150 patients, about 160 patients, about 175patients, about 200 patients, about 250 patients, about 300 patients,about 400 patients or more. In one embodiment, the population of humanstreated is It should be understood that ranges having any one of thesevalues as the upper or lower limits are also intended to be part of thisinvention, e.g., about 10 to about 25, about 15 to about 35, about 25 toabout 50, or about 20 to about 160 patients.

It will be understood that a skilled artisan would be able, uponexamination of one or more art-recognized endpoints, to recognize apatient that had a diminishment of symptoms based upon common knowledgein the art. For example, a skilled artisan would be able to examine andcompare photographs of a skin cancer lesion, such as in situ cutaneoussquamous cell carcinoma, before and after treatment (e.g., such as thephotographs provided herein in the Examples) and be able to recognize adiminishment of symptoms based upon, for example, a diminishment in sizeof the lesion, color of the lesion, or any other visual characteristicof the lesion typically indicative of the cancer. In another example, askilled artisan would be able to examine and compare the tissuepathology of, e.g., a skin cancer, before and after treatment and beable to recognize a diminishment of symptoms based upon a change intissue pathology indicating, e.g., a diminishment in oncogenicity or inseverity of the cancer. In another example, a skilled artisan would beable to examine and compare a CT-scan or MRI image of a tumor or sitesof metastatic lesions before and after treatment, and be able torecognize a diminishment of symptoms based upon, for example, adiminishment in size of a primary tumor or a diminishment in size ornumber of metastatic lesions.

In one embodiment, the amount sufficient to treat the oncologicaldisorder in the human down-regulates anaerobic use of glucose (and/orlactate biosynthesis) and up-regulates mitochondrial oxidativephosphorylation.

In one embodiment, the oncological disorder being treated is not adisorder typically treated via topical administration, e.g., breast orprostate cancer, with the expectation of systemic delivery of an activeagent at therapeutically effective levels.

In one embodiment, the concentration of the env-influencer in thetissues of the human being treated is different than that of a controlstandard of human tissue representative of a healthy or normal state.

In one embodiment, the form of the env-influencer administered to thehuman is different than the predominant form found in systemiccirculation in the human.

In one embodiment, the treatment occurs via an interaction of theenv-influencer with a gene selected from the group of the genes listedin Tables 1-28 (e.g., Tables 2-4, 6-28; especially those genes which up-or down-regulation have been consistently shown in the same cell typesusing different assay methods, or those genes which up- ordown-regulation have been consistently shown across different celltypes, either with the same or different assay methods; preferably themagnitude of up- or down-regulation is identical or similar (e.g., themax fold increase or decrease is no more than 10%, 25%, 50%, 75%, 100%,2-fold, 3-fold, 4-fold, or 5-fold of the min fold increase or decrease).

In one embodiment, the treatment occurs via an interaction of theenv-influencer with a protein selected from the group consisting ofHNF4-alpha, Bcl-xl, Bcl-xS, BNIP-2, Bcl-2, Birc6, Bcl-2-L11 (Bim), XIAP,BRAF, Bax, c-Jun, Bmf, PUMA, cMyc, transaldolase 1, COQ1, COQ3, COQ6,prenyltransferase, 4-hydrobenzoate, neutrophil cytosolic factor 2,nitric oxide synthase 2A, superoxide dismutase 2, VDAC, Bax channel,ANT, Cytochrome c, complex 1, complex II, complex III, complex IV, Foxo3a, DJ-1, IDH-1, Cpt1C, Cam Kinase II, and/or any of the genes listed inTables 1-28 (e.g., Tables 2-4, 6-28).

In one embodiment, the oncological disorder is selected from the groupconsisting: a leukemia, a lymphoma, a melanoma, a carcinoma and asarcoma.

In one embodiment, the method further comprises administering anadditional therapeutic agent or treatment regimen.

In another aspect, the invention provides a method for treating orpreventing an aggressive oncological disorder in a human, comprisingadministering an environmental influencer (env-influencer) to the humanat a selected lower dose than a dosage regimen used or selected for lessaggressive or non-aggressive oncological disorders, thereby treating orpreventing the aggressive oncological disorder.

In a related aspect, the invention provides a method for treating orpreventing a non-aggressive oncological disorder in a human, comprisingadministering an environmental influencer (env-influencer) to the humanat a selected higher dose over a dosage regimen used or selected foraggressive oncological disorders, thereby treating or preventing thenon-aggressive oncological disorder.

In one embodiment, the oncological disorder is selected from the groupconsisting of a leukemia, a lymphoma, a melanoma, a carcinoma and asarcoma.

In one embodiment, the aggressive oncological disorder is selected fromthe group consisting of pancreatic carcinoma, hepatocellular carcinoma,Ewing's sarcoma, metastatic breast cancer, metastatic melanoma, braincancer (astrocytoma, glioblastoma), neuroendocrine cancer, colon cancer,lung cancer, osteosarcoma, androgen-independent prostate cancer, ovariancancer and non-Hodgkin's Lymphoma.

In one embodiment, the non-aggressive oncological disorder is selectedfrom the group consisting of non-metastatic breast cancer,androgen-dependent prostate cancer, small cell lung cancer, acutelymphocytic leukemia.

In one embodiment, the method further comprises a treatment regimenselected from the group consisting of surgery, radiation, hormonetherapy, antibody therapy, therapy with growth factors, cytokines, andchemotherapy.

In yet another aspect, the invention provides a method for (selectively)blocking, in a cancerous cell of a mammal in need of treatment for anoncological disorder, anaerobic use of glucose (glycolysis) andaugmenting mitochondrial oxidative phosphorylation, the methodcomprising: administering to the mammal a therapeutically effectiveamount of at least one env-influencer to selectively block anaerobic useof glucose and to augment mitochondrial oxidative phosphorylation in thecancerous cell of the mammal, towards levels observed in a normal cellof the mammal under normal physiological conditions.

In one embodiment, the method further comprises (1) up-regulating theexpression of one or more genes selected from the group consisting ofthe genes set forth in Tables 1-28 (e.g., 2-4 & 6-28) having a positivefold change; and/or (2) down-regulating the expression of one or moregenes selected from the group consisting of the genes set forth inTables 1-28 (e.g., 2-4 & 6-28) having a negative fold change.

In one embodiment, the method further comprises modulating theexpression of one or more genes selected from the group consisting ofHNF4-alpha, Bcl-xl, Bcl-xS, BNIP-2, Bcl-2, Birc6, Bcl-2-L11 (Bim), XIAP,BRAF, Bax, c-Jun, Bmf, PUMA, cMyc, transaldolase 1, COQ1, COQ3, COQ6,prenyltransferase, 4-hydrobenzoate, neutrophil cytosolic factor 2,nitric oxide synthase 2A, superoxide dismutase 2, VDAC, Bax channel,ANT, Cytochrome c, complex 1, complex II, complex III, complex IV, Foxo3a, DJ-1, IDH-1, Cpt1C and Cam Kinase II.

In one embodiment, the oncological disorder is selected from the groupconsisting of a leukemia, a lymphoma, a melanoma, a carcinoma and asarcoma.

In one embodiment, the method further comprises a treatment regimenselected from the group consisting of surgery, radiation, hormonetherapy, antibody therapy, therapy with growth factors, cytokines, andchemotherapy.

In a still further aspect, the invention provides a method foridentifying an effective environmental influencer for treating,alleviating symptoms of, inhibiting progression of, or preventing anoncological disorder in a mammal, the method comprising: (1) obtaining adiseased biological sample comprising cancer cells of the oncologicaldisorder, and a normal biological sample comprising no cancer cells; (2)contacting the diseased and normal biological samples with a candidateenvironmental influencer; (3) determining the level of expression of oneor more markers present in the diseased and normal biological samples,wherein the marker is selected from the group consisting of the markerslisted in Tables 1-28 (e.g., Tables 2-4 & 6-28) having a positive foldchange and/or having a negative fold change; (4) comparing the level ofexpression of the one of more markers in the diseased and normalbiological samples; wherein an effective environmental influencer isidentified as the candidate environmental influencer that increases thelevel of expression of the one or more markers having a positive foldchange and/or decreases the level of expression of the one or moremarkers having a negative fold change, in the diseased biological samplebut substantially not in the normal biological sample.

In a related aspect, the invention provides a method for treating,alleviating symptoms of, inhibiting progression of, or preventing anoncological disorder in a mammal, the method comprising: (1) obtaining adiseased biological sample comprising cancer cells of the oncologicaldisorder, and a normal biological sample comprising no cancer cells; (2)contacting the diseased and normal biological samples with a candidateenvironmental influencer; (3) determining the level of expression of oneor more markers present in the diseased and normal biological samples,wherein the marker is selected from the group consisting of the markerslisted in Tables 1-28 having a positive fold change and/or having anegative fold change; (4) comparing the level of expression of the oneof more markers in the diseased and normal biological samples; whereinan effective environmental influencer is identified as the candidateenvironmental influencer that increases the level of expression of theone or more markers having a positive fold change and/or decreases thelevel of expression of the one or more markers having a negative foldchange, in the diseased biological sample but substantially not in thenormal biological sample; (5) administering to the mammal the effectiveenvironmental influencer; thereby treating the oncological disorder inthe mammal.

In yet another related embodiment, the invention provides a method foridentifying an effective environmental influencer for treating,alleviating symptoms of, inhibiting progression of, or preventing anoncological disorder in a mammal, the method comprising: (1) obtaining adiseased biological sample comprising cancer cells of the oncologicaldisorder, and a normal biological sample comprising no cancer cells; (2)contacting the diseased and normal biological samples with a candidateenvironmental influencer; (3) determining the level of glycolysis andmitochondrial oxidative phosphorylation in the diseased and normalbiological samples, before and after contacting the candidateenvironmental influencer; wherein an effective environmental influenceris identified as the candidate environmental influencer that increasesthe level of mitochondrial oxidative phosphorylation and/or decreasesthe level of glycolysis, in the diseased biological sample butsubstantially not in the normal biological sample.

In yet another related embodiment, the invention provides a method fortreating, alleviating symptoms of, inhibiting progression of, orpreventing an oncological disorder in a mammal, the method comprising:(1) obtaining a diseased biological sample comprising cancer cells ofthe oncological disorder, and a normal biological sample comprising nocancer cells; (2) contacting the diseased and normal biological sampleswith a candidate environmental influencer; (3) determining the level ofglycolysis and mitochondrial oxidative phosphorylation in the diseasedand normal biological samples, before and after contacting the candidateenvironmental influencer, wherein an effective environmental influenceris identified as the candidate environmental influencer that increasesthe level of mitochondrial oxidative phosphorylation and/or decreasesthe level of glycolysis, in the diseased biological sample butsubstantially not in the normal biological sample; and, (4)administering to the mammal the effective environmental influencer;thereby treating the oncological disorder in the mammal.

In certain embodiments, the level of glycolysis is measured as ECAR,and/or wherein the level of mitochondrial oxidative phosphorylation ismeasured as OCR.

In one embodiment, the env-influencer is not coenzyme Q10.

In a further aspect, the invention provides a method of identifying aMultidimensional Intracellular Molecule (MIM), comprising (a) contactinga cell with an endogenous molecule; (b) monitoring the effect of theendogenous molecule on a cellular microenvironment profile; and (c)identifying an endogenous molecule that induces a change to the cellularmicroenvironment profile; thereby identifying a MIM.

In one embodiment, the method further comprises comparing the effects ofthe endogenous molecule on the cellular microenvironment profile of adiseased cell and a normal control cell; identifying an endogenousmolecule that differentially induces a change to the cellularmicroenvironment profile of the diseased cell as compared to the normalcontrol cell; thereby identifying a MIM.

In one embodiment, the effect on the cellular microenvironment profileis monitored by measuring a change in the level or activity of acellular molecule selected from the group consisting of mRNA, protein,lipid and metabolite.

In another aspect, the invention provides a method of identifying anEpimetabolic shifter (Epi-shifter), comprising (a) comparing molecularprofiles for two or more cells or tissues, wherein the two or more cellsor tissues display differential disease states; (b) identifying amolecule from the moleculer profiles for which a change in levelcorrelates to the disease state; (c) introducing the molecule to a cell;and (d) evaluating the ability of the molecule to shift the metabolicstate of a cell; wherein a molecule capable of shifting the metabolicstate of a cell is identified as an Epi-shifter.

In one embodiment, the molecular profile is selected from the groupconsisting of a metabolite profile, lipid profile, protein profile orRNA profile.

In one embodiment, the molecule does not negatively effect the health orgrowth of a normal cell.

In yet another aspect, the invention provides a method of identifying anagent that is effective in treating an oncological disorder, comprising:(1) providing a candidate environmental influencer; (2) determining theability of the candidate environmental influencer to shift the metabolicstate of a cell; and (3) determining whether the candidate environmentalinfluencer is effective in treating the oncological disorder; whereinthe candidate environmental influencer capable of shifting the metabolicstate of the cell and is effective in treating the oncological disorderis identified as the agent effective in treating the oncologicaldisorder.

In one embodiment, the env-influencer is identified as capable ofshifting the metabolic state of a cell by measuring a change in one ormore of mRNA expression, protein expression, lipid levels, metabolitelevels, levels of bioenergetic molecules, cellular energetics,mitochondrial function and mitochondrial number.

In yet another aspect, the invention provides a composition comprisingan agent identified according to the foregoing methods of the invention.

In another aspect, the invention provides a method for treating,alleviating symptoms of, inhibiting progression of, or preventing aCoQ10 responsive disorder or state in a mammal, the method comprising:administering to the mammal in need there'd a therapeutically effectiveamount of pharmaceutical composition comprising at least oneenvironmental influencer (env-influencer), wherein the environmentalinfluencer selectively elicits, in a disease cell of the mammal, acellular metabolic energy shift towards levels of glycolysis andmitochondrial oxidative phosphorylation observed in a normal cell of themammal under normal physiological conditions.

In certain embodiments, the CoQ10 responsive disorder is an oncologicaldisorder.

Where applicable or not specifically disclaimed, any one of theembodiments described herein are contemplated to be able to combine withany other one or more embodiments, even though the embodiments aredescribed under different aspects of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Sensitivity of SK-MEL-28 to 24 hours of Q10 treatment measuredby the amount of early and late apoptotic cells.

FIG. 2: Sensitivity of SKBR3 to 24 hours of Q10 treatment measured bythe amount of early and late apoptotic cells.

FIG. 3: Sensitivity of PaCa2 to 24 hours of Q10 treatment measured bythe amount of early and late apoptotic cells.

FIG. 4: Sensitivity of PC-3 to 24 hours of Q10 treatment measured by theamount of early and late apoptotic cells.

FIG. 5: Sensitivity of HepG2 to 24 hours of Q10 treatment measured bythe amount of early and late apoptotic cells.

FIG. 6: Sensitivity of MCF-7 to 24 hours of Q10 treatment measured bythe amount of early and late apoptotic cells.

FIG. 7: Measurement of apoptotic cells upon 24 hour treatment with Q10,as measured by Apostrand ELISA method.

FIG. 8: Example gel analysis of 2-D gel electrophoresis. Spots excisedfor identification are marked.

FIG. 9: Network of interaction between proteins identified by 2-D gelelectrophoresis as being modulated by Q10 in SK-MEL-28 cells.

FIG. 10: The pentose phosphate pathway adapted from Verhoeven et al.(Am. J. Hum. Genet. 2001 68(5):1086-1092).

FIG. 11: 2-D gel of the mitochondrial enriched material of SK-MEL-28cells. Spots excised and identified by mass spcectrometrycharacterization are marked.

FIG. 12: Comparative plot of the relative amounts of Q10 present inSK-MEL-28 mitochondria following the exogenous addition of 100 μM Q10into the culture medium.

FIG. 13: Apoptosis pathway mapping known processes.

FIG. 14: Western blot analysis of Bcl-xl.

FIG. 15: Western blot analysis of SK-MEL-28 sample set proved with aVimentin antibody.

FIG. 16: Western blot analysis of cell lysis from a number of celllines, evaluated with five antibodies targeting oxidativephosphorylation complexes (MitoSciences #MS601).

FIG. 17: Western blot comparison of F1-alpha levels.

FIG. 18: Western blot comparison of Q10 response with C-III-Core 2.

FIG. 19: Western blot comparison of Q10 response with C-II-30.

FIG. 20: Western blot comparison of Q10 response with C-IV-COX II.

FIG. 21: Western blot comparison of Q10 response with C-I-20 (ND6).

FIG. 22: Western blot analysis of a variety of cell types against fivemitochondrial protein.

FIG. 23: Western blot comparison of Q10 response with Complex V proteinC-V-α.

FIG. 24: Western blot comparison of Q10 response with C-III-Core 1.

FIG. 25: Western blot comparison of Q10 response with Porin (VDAC1).

FIG. 26: Western blot comparison of Q10 response with Cyclophilin D.

FIG. 27: Western blot comparison of Q10 response with Cytochrome C.

FIG. 28: Theoretical model of Q10 (spheres) inserted into the lipidbinding channel of HNF4alpha (IM7W.pdb) in the Helix 10 openconformation.

FIG. 29: Graph depicting the epidermal CoQ10 concentration in a male pigafter treatment with a composition of the present disclosure having apermeation enhancer.

FIG. 30: Graph depicting the epidermal CoQ10 concentration in a femalepig after treatment with a control composition.

FIG. 31: Photographic depiction of a pre-treated target legion 1.

FIG. 32: Photographic depiction of a post-treated target legion 1.

FIG. 33: Photographic depiction of a pre-treated target legion 2.

FIG. 34: Photographic depiction of a post-treated target legion 2.

FIG. 35: Photographic depiction of a pre-treated target legion 3.

FIG. 36: Photographic depiction of a post-treated target legion 3.

FIG. 37: OCR in HDFa cells in various glucose conditions in normoxic andhypoxic conditions.

FIG. 38: OCR in HASMC cells in various glucose conditions in normoxicand hypoxic conditions.

FIG. 39: OCR values in MCF-7 breast cancer cells in the absence andpresence of 31510 and stressors.

FIG. 40: OCR values in PaCa-2 pancreatic cancer cells in the absence andpresence of 31510 and stressors.

FIG. 41: Graph depicting radioactivity levels in target organs.

FIG. 42: Graph depicting radioactivity levels in waste samples.

FIG. 43: Graph depicting radioactivity levels in blood samples.

DETAILED DESCRIPTION OF THE INVENTION I. Overview and Definitions

As used herein, each of the following terms has the meaning associatedwith it in this section.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e. to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

The term “including” is used herein to mean, and is used interchangeablywith, the phrase “including but not limited to”.

The term “or” is used herein to mean, and is used interchangeably with,the term “and/or,” unless context clearly indicates otherwise.

The term “such as” is used herein to mean, and is used interchangeably,with the phrase “such as but not limited to”.

A “patient” or “subject” to be treated by the method of the inventioncan mean either a human or non-human animal, preferably a mammal. Itshould be noted that clinical observations described herein were madewith human subjects and, in at least some embodiments, the subjects arehuman.

“Therapeutically effective amount” means the amount of a compound that,when administered to a patient for treating a disease, is sufficient toeffect such treatment for the disease. When administered for preventinga disease, the amount is sufficient to avoid or delay onset of thedisease. The “therapeutically effective amount” will vary depending onthe compound, the disease and its severity and the age, weight, etc., ofthe patient to be treated.

“Preventing” or “prevention” refers to a reduction in risk of acquiringa disease or disorder (i.e., causing at least one of the clinicalsymptoms of the disease not to develop in a patient that may be exposedto or predisposed to the disease but does not yet experience or displaysymptoms of the disease).

The term “prophylactic” or “therapeutic” treatment refers toadministration to the subject of one or more of the subjectcompositions. If it is administered prior to clinical manifestation ofthe unwanted condition (e.g., disease or other unwanted state of thehost animal) then the treatment is prophylactic, i.e., it protects thehost against developing the unwanted condition, whereas if administeredafter manifestation of the unwanted condition, the treatment istherapeutic (i.e., it is intended to diminish, ameliorate or maintainthe existing unwanted condition or side effects therefrom).

The term “therapeutic effect” refers to a local or systemic effect inanimals, particularly mammals, and more particularly humans caused by apharmacologically active substance. The term thus means any substanceintended for use in the diagnosis, cure, mitigation, treatment orprevention of disease or in the enhancement of desirable physical ormental development and conditions in an animal or human. The phrase“therapeutically-effective amount” means that amount of such a substancethat produces some desired local or systemic effect at a reasonablebenefit/risk ratio applicable to any treatment. In certain embodiments,a therapeutically-effective amount of a compound will depend on itstherapeutic index, solubility, and the like. For example, certaincompounds discovered by the methods of the present invention may beadministered in a sufficient amount to produce a reasonable benefit/riskratio applicable to such treatment.

By “patient” is meant any animal (e.g., a human or a non-human mammal)that can be subjected to at least one medical intervention (e.g.,treatment, diagnostic/prognostic tests, etc.), including horses, dogs,cats, pigs, goats, rabbits, hamsters, monkeys, guinea pigs, rats, mice,lizards, snakes, sheep, cattle, fish, and birds.

“Metabolic pathway” refers to a sequence of enzyme-mediated reactionsthat transform one compound to another and provide intermediates andenergy for cellular functions. The metabolic pathway can be linear orcyclic.

“Metabolic state” refers to the molecular content of a particularcellular, multicellular or tissue environment at a given point in timeas measured by various chemical and biological indicators as they relateto a state of health or disease.

The term “microarray” refers to an array of distinct polynucleotides,oligonucleotides, polypeptides (e.g., antibodies) or peptidessynthesized on a substrate, such as paper, nylon or other type ofmembrane, filter, chip, glass slide, or any other suitable solidsupport.

The terms “disorders” and “diseases” are used inclusively and refer toany deviation from the normal structure or function of any part, organor system of the body (or any combination thereof). A specific diseaseis manifested by characteristic symptoms and signs, includingbiological, chemical and physical changes, and is often associated witha variety of other factors including, but not limited to, demographic,environmental, employment, genetic and medically historical factors.Certain characteristic signs, symptoms, and related factors can bequantitated through a variety of methods to yield important diagnosticinformation.

The term “expression” is used herein to mean the process by which apolypeptide is produced from DNA. The process involves the transcriptionof the gene into mRNA and the translation of this mRNA into apolypeptide. Depending on the context in which used, “expression” mayrefer to the production of RNA, protein or both.

The terms “level of expression of a gene” or “gene expression level”refer to the level of mRNA, as well as pre-mRNA nascent transcript(s),transcript processing intermediates, mature mRNA(s) and degradationproducts, or the level of protein, encoded by the gene in the cell.

The term “modulation” refers to upregulation (i.e., activation orstimulation), downregulation (i.e., inhibition or suppression) of aresponse, or the two in combination or apart. A “modulator” is acompound or molecule that modulates, and may be, e.g., an agonist,antagonist, activator, stimulator, suppressor, or inhibitor.

The term “intermediate of the coenzyme biosynthesis pathway” as usedherein, characterizes those compounds that are formed between thechemical/biological conversion of tyrosine and Acetyl-CoA to uqiquinone.Intermediates of the coenzyme biosynthesis pathway include3-hexaprenyl-4-hydroxybenzoate, 3-hexaprenyl-4,5-dihydroxybenzoate,3-hexaprenyl-4-hydroxy-5-methoxybenzoate,2-hexaprenyl-6-methoxy-1,4-benzoquinone,2-hexaprenyl-3-methyl-6-methoxy-1,4-benzoquinone,2-hexaprenyl-3-methyl-5-hydroxy-6-methoxy-1,4-benzoquinone,3-Octaprenyl-4-hydroxybenzoate, 2-octaprenylphenol,2-octaprenyl-6-metholxyphenol,2-octaprenyl-3-methyl-6-methoxy-1,4-benzoquinone,2-octaprenyl-3-methyl-5-hydroxy-6-methoxy-1,4-benzoquinone,2-decaprenyl-3-methyl-5-hydroxy-6-methoxy-1,4-benzoquinone,2-decaprenyl-3-methyl-6-methoxy-1,4-benzoquinone,2-decaprenyl-6-methoxy-1,4-benzoquinone, 2-decaprenyl-6-methoxyphenol,3-decaprenyl-4-hydroxy-5-methoxybenzoate,3-decaprenyl-4,5-dihydroxybenzoate, 3-decaprenyl-4-hydroxybenzoate,4-hydroxy phenylpyruvate, 4-hydroxyphenyllactate, 4-hydroxybenzoate,4-hydroxycinnamate and hexaprenydiphosphate.

The term “Trolamine,” as used herein, refers to Trolamine NF,Triethanolamine, TEAlan®, TEAlan 99%, Triethanolamine, 99%,Triethanolamine, NF or Triethanolamine, 99%, NF. These terms may be usedinterchangeably herein.

In some embodiments, the compounds of the present invention, e.g., theMIMs or epi-shifters described herein, may be used to treat a CoenzymeQ10 responsive state in a subject in need thereof. The language“Coenzyme Q10 responsive state,” or “CoQ10 responsive state/disease,”includes diseases, disorders, states and/or conditions which can betreated, prevented, or otherwise ameliorated by the administration ofCoenzyme Q10. Without wishing to be bound by any particular theory, andas described further herein, it is believed that CoQ10 functions, atleast partially, by inducing a metabolic shift to the cellmicroenvironment, such as a shift towards the type and/or level ofoxidative phosphorylation in normal state cells. Accordingly, in someembodiments, CoQ10 responsive states are states that arise from analtered metabolism of cell microenvironment. Coenzyme Q10 responsivestates include, for example, oncological disorders, which, for example,may be biased towards glycolysis and lactate biosynthesis. In someembodiments, CoQ10 responsive oncological disorders include livercancer, pancreatic cancer, breast cancer, prostate cancer, liver cancer,or bone cancer, squamous cell carcinomas, basal cell carcinomas,melanomas, and actinic keratosis, among others. Coenzyme Q10 responsivestates further include other oncological disorders as described herein.

Coenzyme Q10 responsive states also include, for example, metabolicdisorders such as obesity, diabetes, pre-diabetes, Metabolic Syndrome,satiety, and endocrine abnormalities. Coenzyme Q10 responsive statesfurther include other metabolic disorders as described herein.

In some embodiments, the compounds of the present invention, e.g., theMIMs or epi-shifters described herein, share a common activity withCoenzyme Q10. As used herein, the phrase “share a common activity withCoenzyme Q10” refers to the ability of a compound to exhibit at least aportion of the same or similar activity as Coenzyme Q10. In someembodiments, the compounds of the present invention exhibit 25% or moreof the activity of Coenzyme Q10. In some embodiments, the compounds ofthe present invention exhibit up to and including about 130% of theactivity of Coenzyme Q10. In some embodiments, the compounds of thepresent invention exhibit about 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%,38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%,52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%,66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%,107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%,119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, or130% of the activity of Coenzyme Q10. It is to be understood that eachof the values listed in this paragraph may be modified by the term“about.” Additionally, it is to be understood that any range which isdefined by any two values listed in this paragraph is meant to beencompassed by the present invention. For example, in some embodiments,the compounds of the present invention exhibit between about 50% andabout 100% of the activity of Coenzyme Q10. In some embodiments, theactivity shared by Coenzyme Q10 and the compounds of the presentinvention is the ability to induce a shift in cellular metabolism. Incertain embodiments, the activity shared by of CoQ10 and the compoundsof the present invention is measured by OCR (Oxygen Consumption Rate)and/or ECAR (ExtraCellular Acidification Rate).

As used herein, “oncological disorder” refers to all types of cancer orneoplasm or malignant tumors found in humans, including, but not limitedto: leukemias, lymphomas, melanomas, carcinomas and sarcomas. As usedherein, the terms or language “oncological disorder”, “cancer,”“neoplasm,” and “tumor,” are used interchangeably and in either thesingular or plural form, refer to cells that have undergone a malignanttransformation that makes them pathological to the host organism. Insome embodiments the oncological disorder is a Coenzyme Q10 responsivestate.

In some embodiments, the oncological disorder or cancer is characterizedby a lack of apoptosis. In other embodiments, the oncological disorderor cancer is characterized by increased angiogenesis. In otherembodiments, the oncological disorder or cancer is characterized byextracellular matrix (ECM) degradation. In yet other embodiments, theoncological disorder or cancer is characterized by loss of cell cyclecontrol. In still other embodiments, the oncological disorder or canceris characterized by a shift in metabolic governance from mitochondrialoxidative phosphorylation to increased utilization and/or dependency onlactate and glycolytic flux. In further embodiments, the oncologicaldisorder or cancer is characterized by adapted immunomodulatorymechanisms that have evaded immunosurveilance. In one embodiment, theoncological disorder or cancer is characterized by at least two of theabove features, e.g., increased angiogenesis and ECM degradation. In oneembodiment, the oncological disorder or cancer is characterized by atleast three of the above features. In one embodiment, the oncologicaldisorder or cancer is characterized by at least four of the abovefeatures. In one embodiment, the oncological disorder or cancer ischaracterized by at least five of the above features. In one embodiment,the oncological disorder or cancer is characterized by all six of theabove features.

Accordingly, in some embodiments, the compounds of the present inventionfunction by restoring the capacity for apoptosis or inducing apoptosis.In other embodiments, the compounds of the present invention function byreducing, decreasing or inhibiting angiogenesis. In still otherembodiments, the compounds of the present invention function byrestoring re-establishing extracellular matrix. In other embodiments,the compounds of the present invention function by restoring cell cyclecontrol. In still other embodiments, the compounds of the presentinvention function by shifting metabolic governance back from glycolysisto mitochondrial oxidative phosphorylation. In further embodiments, thecompounds of the present invention function by restoringimmuno-surveillance or restoring the body's ability to recognize thecancer cell as foreign.

Without wishing to be bound by any particular theory, it is believedthat there is typically a coordinated cascade of events that aggregateto develop into cancer. That is, in some embodiments, cancer is notsingularly dependent on a 1 gene-1 protein-root causality. In someembodiments, cancer is a physiologic disease state that manifests intotissue changes and alterations that become tumors, altered tissuestates, e.g., energetics, compromised extracellular matrix integritythat allows for metastatic potential, lack of immunosurveilance and/oraltered state of angiogenesis.

Primary cancer cells (that is, cells obtained from near the site ofmalignant transformation) can be readily distinguished fromnon-cancerous cells by well-established techniques, particularlyhistological examination. The definition of a cancer cell, as usedherein, includes not only a primary cancer cell, but also cancer stemcells, as well as cancer progenitor cells or any cell derived from acancer cell ancestor. This includes metastasized cancer cells, and invitro cultures and cell lines derived from cancer cells. When referringto a type of cancer that normally manifests as a solid tumor, a“clinically detectable” tumor is one that is detectable on the basis oftumor mass; e.g., by procedures such as CAT scan, MR imaging, X-ray,ultrasound or palpation, and/or which is detectable because of theexpression of one or more cancer-specific antigens in a sampleobtainable from a patient.

As used herein, “positive fold change” refers to “up-regulation” or“increase (of expression)” of a gene that is listed in the relevanttables.

As used herein, “negative fold change” refers to “down-regulation” or“decrease (of expression)” of a gene that is listed in the relevanttables.

In certain embodiments, where a particular listed gene is associatedwith more than one treatment conditions, such as at different timeperiods after a treatment, or treatment by different concentrations of apotential environmental influencer (e.g., CoQ10), the fold change forthat particular gene refers to the longest recorded treatment time. Inother embodiments, the fold change for that particular gene refers tothe shortest recorded treatment time. In other embodiments, the foldchange for that particular gene refers to treatment by the highestconcentration of env-influencer (e.g., CoQ10). In other embodiments, thefold change for that particular gene refers to treatment by the lowestconcentration of env-influencer (e.g., CoQ10). In yet other embodiments,the fold change for that particular gene refers to the modulation (e.g.,up- or down-regulation) in a manner that is consistent with thetherapeutic effect of the env-influencer.

In certain embodiments, the positive or negative fold change refers tothat of any gene listed in any of the Tables 1-28 (e.g., 2-4 & 6-28). Incertain embodiments, the positive or negative fold change refers to thatof any gene listed in any of the Tables 1-28 (e.g., 2-4 & 6-28), exceptfor one of the tables (e.g., except for Table 1, except for Table 5,etc.). In certain embodiments, the positive or negative fold changerefers to that of any gene listed in any of the Tables 1-28 (e.g., 2-4 &6-28), except for any two of the tables (e.g., except for Tables 1 and5, except for Table 2 & 16, etc.). In certain embodiments, the positiveor negative fold change refers to that of any gene listed in any of theTables 1-28 (e.g., 2-4 & 6-28), except for any three of the tables; orexcept for any four of the tables; or except for any 5, 6, 7, 8, 9, 10,or more of the tables. In certain embodiments, the positive or negativefold change refers to that of any gene listed in any of the Tables 1-28(e.g., 2-4 & 6-28), except for tables 1, 5, 9, and 12.

Reference will now be made in detail to preferred embodiments of theinvention. While the invention will be described in conjunction with thepreferred embodiments, it will be understood that it is not intended tolimit the invention to those preferred embodiments. To the contrary, itis intended to cover alternatives, modifications, and equivalents as maybe included within the spirit and scope of the invention as defined bythe appended claims.

II. Environmental Influencers

The present invention provides methods of treating oncological disordersby administration of an Environmental influencer. “Environmentalinfluencers” (Env-influencers) are molecules that influence or modulatethe disease environment of a human or a non-human mammal in a beneficialmanner allowing the human's (or the non-human mammal's) diseaseenvironment to shift, reestablish back to or maintain a normal orhealthy environment leading to a normal state. Env-influencers includeboth Multidimensional Intracellular Molecules (MIMs) and Epimetabolicshifters (Epi-shifters) as defined below.

In certain embodiments, the MIMS and Epi-shifters disclosed hereinexclude those that are conventionally used as a dietary supplement. Incertain embodiments, these MIMS and/or Epi-shifter that are disclosedherein are of pharmaceutical grade. In certain embodiments, the MIMSand/or Epi-shifter of pharmaceutical grade has a purity between about95% and about 100% and include all values between 95% and 100%. Incertain embodiments, the purity of the MIMS and/or Epi-shifter is 95%,96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%,99.8%, 99.9%, 99.9 or 100%. In certain embodiments, the MIMS and/orEpi-shifter is free or substantially free of endotoxins. In otherembodiments, the MIMS and/or Epi-shifter is free or substantially freeof foreign protein materials. In certain embodiments, the MIMS and/orEpi-shifter is CoQ10.

1. Multidimensional Intracellular Molecule (MIM)

The term “Multidimensional Intracellular Molecule (MIM)”, is an isolatedversion or synthetically produced version of an endogenous molecule thatis naturally produced by the body and/or is present in at least one cellof a human. A MIM is characterized by one or more, two or more, three ormore, or all of the following functions. MIMs are capable of entering acell, and the entry into the cell includes complete or partial entryinto the cell, as long as the biologically active portion of themolecule wholly enters the cell. MIMs are capable of inducing a signaltransduction and/or gene expression mechanism within a cell. MIMs aremultidimensional in that the molecules have both a therapeutic and acarrier, e.g., drug delivery, effect. MIMs also are multidimensional inthat the molecules act one way in a disease state and a different way ina normal state. For example, in the case of CoQ-10, administration ofCoQ-10 to a melanoma cell in the presence of VEGF leads to a decreasedlevel of Bcl2 which, in turn, leads to a decreased oncogenic potentialfor the melanoma cell. In contrast, in a normal fibroblast,co-administration of CoQ-10 and VEFG has no effect on the levels ofBcl2. Preferably, MIMs selectively act in cells of a disease state, andhave substantially no effect in (matching) cells of a normal state.Preferably, MIMs selectively renders cells of a disease state closer inphenotype, metabolic state, genotype, mRNA/protein expression level,etc. to (matching) cells of a normal state.

In one embodiment, a MIM is also an epi-shifter. In another embodiment,a MIM is not an epi-shifter. The skilled artisan will appreciate that aMIM of the invention is also intended to encompass a mixture of two ormore endogenous molecules, wherein the mixture is characterized by oneor more of the foregoing functions. The endogenous molecules in themixture are present at a ratio such that the mixture functions as a MIM.

MIMs can be lipid based or non-lipid based molecules. Examples of MIMsinclude, but are not limited to, CoQ10, acetyl Co-A, palmityl Co-A,L-carnitine, amino acids such as, for example, tyrosine, phenylalanine,and cysteine. In one embodiment, the MIM is a small molecule. In oneembodiment of the invention, the MIM is not CoQ10. MIMs can be routinelyidentified by one of skill in the art using any of the assays describedin detail herein.

In some embodiments, MIMs include compounds in the Vitamin B family, ornucleosides, mononucleotides or dinucleotides that comprise a compoundin the Vitamin B family. Compounds in the vitamin B family include, forexample, thiamine (vitamin B1), niacin (also known as nicotinic acid orVitamin B3), or pyridoxine (vitamin B6) as well as provitamins such aspanthenol (provitamin B5). In some embodiments, the MIM is selected fromthiamine, niacin and pyridoxine. Nucleosides, mononucleotides ordinucleotides that comprise a compound in the vitamin B family include,for example, nucleosides, mononucleotides or dinucleotides which includean adenine or a niacin (nicotinic acid) molecule. In some embodiments,the MIM is selected from adenosine, adenosine diphosphate (ADP), flavinadenosine dinucleotide (FAD, which comprises parts of vitamin B2 andADP) and nicotinic acid dinucleotide.

In other embodiments, the MIMs include amino acids. Examples of aminoacids include, for example, tyrosine (e.g., L-tyrosine), cysteine,phenylalanine (e.g., L-phenylalanine) and alanine. In some embodiments,the amino acid is phenylalanine or alanine. In some embodiments, theMIMs include amino acid derivatives such as 4-hydroxyphenylpyruvate oracetylglycine.

In some embodiment, the MIM is a glucose analog, e.g., a glucosemolecule wherein one —OH or —CH₂OH substituent has been replaced with a—COOH, a —COO⁻ or an —NH₂ substituent. Examples of glucose analogsinclude glucosamine, glucuronic acid, glucuronide and glucuronate.

In some embodiments, the MIM is selected from compounds of formula (I):

-   -   wherein    -   n is an integer of 0 or 1;    -   R¹, R², R³ and R⁴, when present, are each independently selected        from hydrogen and hydroxyl or R¹ and R² are taken together with        the carbon on which they are attached to form a carbonyl (C═O)        group;    -   W is —COOH or —N(CH₃)₃ ⁺; and    -   X is hydrogen, a negative charge or a alkali metal cation, such        as Na⁺ or.    -   It is to be understood that when n is 0, the CHR³ group is        bonded to the W substituent.

In some embodiments, W is —N(CH₃)₃ ⁺. In some embodiments, the MIM is acarnitine, such as L-carnitine.

In some embodiments, the MIM is a dicarboxylic acid. In someembodiments, W is —COOH. In some embodiments, R³ is hydrogen. In someembodiments, n is 0. In some embodiments, R¹ and R² are eachindependently hydrogen. In some embodiments, W is —COOH, R³ is hydrogen,n is 0 and R¹ and R² are each independently hydrogen. In someembodiments, n is 1. In some embodiments R¹ and R² are taken togetherwith the carbon on which they are attached to form a carbonyl (C═O)group. In some embodiments, R⁴ is hydrogen. In some embodiments, R⁴ ishydroxyl. In some embodiments, W is —COOH, R³ is hydrogen, n is 1 and R¹and R² are taken together with the carbon on which they are attached toform a carbonyl (C═O) group.

In some embodiments, the MIM is an intermediate of the Krebs Cycle, theexcess of which drives the Krebs Cycle towards productive oxidativephosphorylation. Exemplary Krebs Cycle intermediates that are MIMsinclude succinic acid or succinate, malic acid or malate, andα-ketoglutaric acid or α-ketoglutarate.

In some embodiments, the MIM is a building block of CoQ10, which has thefollowing structure:

Thus, building blocks of CoQ10 include, but are not limited to,phenylalanine, tyrosine, 4-hydroxyphenylpyruvate, phenylacetate,3-methoxy-4-hydroxymandelate, vanillic acid, 4-hydroxybenzoate,mevalonic acid, farnesyl, 2,3-dimethoxy-5-methyl-p-benzoquinone, as wellas the corresponding acids or ions thereof. In some embodiments, the MIMis selected from phenylalanine, tyrosine, 4-hydroxyphenylpyruvate,phenylacetate and 4-hydroxybenzoate.

(i) Methods of Identifying MIMS

The present invention provides methods for identifying a MIM. Methodsfor identifying a MIM involve, generally, the exogenous addition to acell of an endogenous molecule and evaluating the effect on the cell,e.g., the cellular microenvironment profile, that the endogenousmolecule provides. Effects on the cell are evaluated at one or more ofthe cellular, mRNA, protein, lipid, and/or metabolite level to identifyalterations in the cellular microenvironment profile. In one embodiment,the cells are cultured cells, e.g., in vitro. In one embodiment, thecells are present in an organism. The endogenous molecule may be addedto the cell at a single concentration or may be added to the cell over arange of concentrations. In one embodiment, the endogenous molecule isadded to the cells such that the level of the endogenous molecule in thecells is elevated (e.g., is elevated by 1.1 fold, 1.2 fold, 1.3 fold,1.4 fold, 1.5 fold, 1.6 fold, 1.7 fold, 1.8 fold, 1.9 fold, 2.0 fold,3.0 fold, 4.0 fold, 5.0 fold, 10 fold, 15 fold, 20 fold, 25 fold, 30fold, 35 fold, 40 fold, 45 fold, 50 fold or greater) as compared to thelevel of the endogenous molecule in a control, untreated cell.

Molecules that induce a change in the cell as detected by alterationsin, for example, any one or more of morphology, physiology, and/orcomposition (e.g., mRNA, protein, lipid, metabolite) may be evaluatedfurther to determine if the induced changes to the cellularmicroenvironment profile are different between a disease cellular stateand a normal cellular state. Cells (e.g., cell culture lines) of diversetissue origin, cell type, or disease state may be evaluated forcomparative evaluation. For example, changes induced in the cellularmicroenvironment profile of a cancer cell may be compared to changesinduced to a non-cancerous or normal cell. An endogenous molecule thatis observed to induce a change in the microenvironment profile of a cell(e.g., induces a change in the morphology, physiology and/orcomposition, e.g., mRNA, protein, lipid or metabolite, of the cell)and/or to differentially (e.g., preferentially) induce a change in themicroenvironment profile of a diseased cell as compared to a normalcell, is identified as a MIM.

MIMs of the invention may be lipid based MIMs or non-lipid based MIMs.Methods for identifying lipid based MIMs involve the above-describedcell based methods in which a lipid based endogenous molecule isexogenously added to the cell. In a preferred embodiment, the lipidbased endogenous molecule is added to the cell such that the level ofthe lipid based endogenous molecule in the cell is elevated. In oneembodiment, the level of the lipid based endogenous molecule is elevatedby 1.1 fold, 1.2 fold, 1.3 fold, 1.4 fold, 1.5 fold, 1.6 fold, 1.7 fold,1.8 fold, 1.9 fold, 2.0 fold, 3.0 fold, 4.0 fold, 5.0 fold, 10 fold, 15fold, 20 fold, 25 fold, 30 fold, 35 fold, 40 fold, 45 fold, 50 fold orgreater as compared to the level in an untreated control cell.Formulation and delivery of the lipid based molecule to the cell isdependent upon the properties of each molecule tested, but many methodsare known in the art. Examples of formulation and delivery of lipidbased molecules include, but are not limited to, solubilization byco-solvents, carrier molecules, liposomes, dispersions, suspensions,nanoparticle dispersions, emulsions, e.g., oil-in-water or water-in-oilemulsions, multiphase emulsions, e.g., oil-in-water-in-oil emulsions,polymer entrapment and encapsulation. The delivery of the lipid basedMIM to the cell can be confirmed by extraction of the cellular lipidsand quantification of the MIM by routine methods known in the art, suchas mass spectrometry.

Methods for identifying non-lipid based MIMs involve the above-describedcell based methods in which a non-lipid based endogenous molecule isexogenously added to the cell. In a preferred embodiment, the non-lipidbased endogenous molecule is added to the cell such that the level ofthe non-lipid based endogenous molecule in the cell is elevated. In oneembodiment, the level of the non-lipid based endogenous molecule iselevated by 1.1 fold, 1.2 fold, 1.3 fold, 1.4 fold, 1.5 fold, 1.6 fold,1.7 fold, 1.8 fold, 1.9 fold, 2.0 fold, 3.0 fold, 4.0 fold, 5.0 fold, 10fold, 15 fold, 20 fold, 25 fold, 30 fold, 35 fold, 40 fold, 45 fold, 50fold or greater as compared to the level in an untreated control cell.Formulation and delivery of the non-lipid based molecule to the cell isdependent upon the properties of each molecule tested, but many methodsare known in the art. Examples of formulations and modes of delivery ofnon-lipid based molecules include, but are not limited to,solubilization by co-solvents, carrier molecules, active transport,polymer entrapment or adsorption, polymer grafting, liposomalencapsulation, and formulation with targeted delivery systems. Thedelivery of the non-lipid based MIM to the cell may be confirmed byextraction of the cellular content and quantification of the MIM byroutine methods known in the art, such as mass spectrometry.

2. Epimetabolic Shifters (Epi-Shifters)

As used herein, an “epimetabolic shifter” (epi-shifter) is a molecule(endogenous or exogenous) that modulates the metabolic shift from ahealthy (or normal) state to a disease state and vice versa, therebymaintaining or reestablishing cellular, tissue, organ, system and/orhost health in a human. Epi-shifters are capable of effectuatingnormalization in a tissue microenvironment. For example, an epi-shifterincludes any molecule which is capable, when added to or depleted from acell, of affecting the microenvironment (e.g., the metabolic state) of acell. The skilled artisan will appreciate that an epi-shifter of theinvention is also intended to encompass a mixture of two or moremolecules, wherein the mixture is characterized by one or more of theforegoing functions. The molecules in the mixture are present at a ratiosuch that the mixture functions as an epi-shifter. Examples ofepi-shifters include, but are not limited to, coQ-10; vitamin D3; ECMcomponents such as fibronectin; immunomodulators, such as TNFα or any ofthe interleukins, e.g., IL-5, IL-12, IL-23; angiogenic factors; andapoptotic factors.

In some embodiments, the epi-shifter is an enzyme, such as an enzymethat either directly participates in catalyzing one or more reactions inthe Krebs Cycle, or produces a Krebs Cycle intermediate, the excess ofwhich drive the Krebs Cycle. In some embodiments, the enzyme is anenzyme of the non-oxidative phase of the pentose phosphate pathway, suchas transaldolase, or transketolase. In other embodiments, the enzyme isa component enzyme or enzyme complex that facilitates the Krebs Cycle,such as a synthase or a ligase. Exemplary enzymes include succinyl CoAsynthase (Krebs Cycle enzyme) or pyruvate carboxylase (a ligase thatcatalyzes the reversible carboxylation of pyruvate to form oxaloacetate(OAA), a Krebs Cycle intermediate).

In some embodiments, the epi-shifter is a building block of CoQ10.Building blocks of CoQ10 include, but are not limited to, phenylalanine,tyrosine, 4-hydroxyphenylpyruvate, phenylacetate,3-methoxy-4-hydroxymandelate, vanillic acid, 4-hydroxybenzoate,mevalonic acid, farnesyl, 2,3-dimethoxy-5-methyl-p-benzoquinone, as wellas the corresponding acids or ions thereof. In some embodiments, theepi-shifter is selected from phenylalanine, tyrosine,4-hydroxyphenylpyruvate, phenylacetate and 4-hydroxybenzoate.

In some embodiments, the epi-shifter is a compound in the Vitamin Bfamily. Compounds in the vitamin B family include, for example,riboflavin (vitamin B2), or analogs thereof. Epi-shifters also includeany analogs or pro-drugs that may be metabolized in vivo to any of theendogenous MIMs, such as those described herein.

In one embodiment, the epi-shifter also is a MIM. In one embodiment, theepi-shifter is not CoQ10. Epi-shifters can be routinely identified byone of skill in the art using any of the assays described in detailherein.

(i) Methods of Identifying Epi-Shifters

Epimetabolic shifters (epi-shifter) are molecules capable of modulatingthe metabolic state of a cell, e.g., inducing a metabolic shift from ahealthy (or normal) state to a disease state and vice versa, and arethereby capable of maintaining or reestablishing cellular, tissue,organ, system and/or host health in a human. Epi-shifters of theinvention thus have utility in the diagnostic evaluation of a diseasedstate. Epi-shifters of the invention have further utility in therapeuticapplications, wherein the application or administration of theepi-shifter (or modulation of the epi-shifter by other therapeuticmolecules) effects a normalization in a tissue microenvironment and thedisease state.

The identification of an epimetabolic shifter involves, generally,establishing a molecular profile, e.g., of metabolites, lipids, proteinsor RNAs (as individual profiles or in combination), for a panel of cellsor tissues that display differential disease states, progression, oraggressiveness A molecule from the profile(s) for which a change inlevel (e.g., an increased or decreased level) correlates to the diseasestate, progression or aggressiveness is identified as a potentialepi-shifter.

In one embodiment, an epi-shifter is also a MIM. Potential epi-shiftersmay be evaluated for their ability to enter cells upon exogenousaddition to a cell by using any number of routine techniques known inthe art, and by using any of the methods described herein. For example,entry of the potential epi-shifter into a cell may be confirmed byextraction of the cellular content and quantification of the potentialepi-shifter by routine methods known in the art, such as massspectrometry. A potential epi-shifter that is able to enter a cell isthereby identified as a MIM.

To identify an epi-shifter, a potential epi-shifter is next evaluatedfor the ability to shift the metabolic state of a cell. The ability of apotential epi-shifters to shift the metabolic state of the cellmicroenvironment is evaluated by introducing (e.g., exogenously adding)to a cell a potential epi-shifter and monitoring in the cell one or moreof: changes in gene expression (e.g., changes in mRNA or proteinexpression), concentration changes in lipid or metabolite levels,changes in bioenergetic molecule levels, changes in cellular energetics,and/or changes in mitochondrial function or number. Potentialepi-shifters capable of shifting the metabolic state of the cellmicroenvironment can be routinely identified by one of skill in the artusing any of the assays described in detail herein. Potentialepi-shifters are further evaluated for the ability to shift themetabolic state of a diseased cell towards a normal healthy state (orconversely, for the ability to shift the metabolic state of a normalcell towards a diseased state). A potential epi-shifter capable ofshifting the metabolic state of a diseased cell towards a normal healthystate (or of shifting the metabolic state of healthy normal cell towardsa diseased state) is thus identified as an Epi-shifter. In a preferredembodiment, the epi-shifter does not negatively impact the health and/orgrowth of normal cells.

Epimetabolic shifters of the invention include, but are not limited to,small molecule metabolites, lipid-based molecules, and proteins andRNAs. To identify an epimetabolic shifter in the class of small moleculeendogenous metabolites, metabolite profiles for a panel of cells ortissues that display differential disease states, progression, oraggressiveness are established. The metabolite profile for each cell ortissue is determined by extracting metabolites from the cell or tissueand then identifying and quantifying the metabolites using routinemethods known to the skilled artisan, including, for example,liquid-chromatography coupled mass spectrometry or gas-chromatographycouple mass spectrometry methods. Metabolites for which a change inlevel (e.g., an increased or decreased level) correlates to the diseasestate, progression or aggressiveness, are identified as potentialepi-shifters.

To identify epimetabolic shifters in the class of endogenous lipid-basedmolecules, lipid profiles for a panel of cells or tissues that displaydifferential disease states, progression, or aggressiveness areestablished. The lipid profile for each cell or tissue is determined byusing lipid extraction methods, followed by the identification andquantitation of the lipids using routine methods known to the skilledartisan, including, for example, liquid-chromatography coupled massspectrometry or gas-chromatography couple mass spectrometry methods.Lipids for which a change in level (e.g., an increase or decrease inbulk or trace level) correlates to the disease state, progression oraggressiveness, are identified as potential epi-shifters.

To identify epimetabolic shifters in the class of proteins and RNAs,gene expression profiles for a panel of cells or tissues that displaydifferential disease states, progression, or aggressiveness areestablished. The expression profile for each cell or tissue isdetermined at the mRNA and/or protein level(s) using standard proteomic,mRNA array, or genomic array methods, e.g., as described in detailherein. Genes for which a change in expression (e.g., an increase ordecrease in expression at the mRNA or protein level) correlates to thedisease state, progression or aggressiveness, are identified aspotential epi-shifters.

Once the molecular profiles described above are established (e.g., forsoluble metabolites, lipid-based molecules, proteins, RNAs, or otherbiological classes of composition), cellular and biochemical pathwayanalysis is carried out to elucidate known linkages between theidentified potential epi-shifters in the cellular environment. Thisinformation obtained by such cellular and/or biochemical pathwayanalysis may be utilized to categorize the pathways and potentialepi-shifters.

The utility of an Epi-shifter to modulate a disease state can be furtherevaluated and confirmed by one of skill in the art using any number ofassays known in the art or described in detail herein. The utility of anEpi-shifter to modulate a disease state can be evaluated by directexogenous delivery of the Epi-shifter to a cell or to an organism. Theutility of an Epi-shifter to modulate a disease state can alternativelybe evaluated by the development of molecules that directly modulate theEpi-shifter (e.g., the level or activity of the Epi-shifter). Theutility of an Epi-shifter to modulate a disease state can also beevaluated by the development of molecules that indirectly modulate theEpi-shifter (e.g., the level or activity of the Epi-shifter) byregulating other molecules, such as genes (e.g., regulated at the RNA orprotein level), placed in the same pathway as the Epi-shifter.

The Epimetabolomic approach described herein facilitates theidentification of endogenous molecules that exist in a cellularmicroenvironment and the levels of which are sensed and controlledthrough genetic, mRNA, or protein-based mechanisms. The regulationresponse pathways found in normal cells that are triggered by anEpi-shifter of the invention may provide a therapeutic value in amisregulated or diseased cellular environment. In addition, theepimetabolic approach described herein identifies epi-shifters that mayprovide a diagnostic indication for use in clinical patient selection, adisease diagnostic kit, or as a prognostic indicator.

III. Assays Useful for Identifying MIMs/Epi-Shifters

Techniques and methods of the present invention employed to separate andidentify molecules and compounds of interest include but are not limitedto: liquid chromatography (LC), high-pressure liquid chromatography(HPLC), mass spectroscopy (MS), gas chromatography (GC), liquidchromatography/mass spectroscopy (LC-MS), gas chromatography/massspectroscopy (GC-MS), nuclear magnetic resonance (NMR), magneticresonance imaging (MRI), Fourier Transform InfraRed (FT-IR), andinductively coupled plasma mass spectrometry (ICP-MS). It is furtherunderstood that mass spectrometry techniques include, but are notlimited to, the use of magnetic-sector and double focusing instruments,transmission quadrapole instruments, quadrupole ion-trap instruments,time-of-flight instruments (TOF), Fourier transform ion cyclotronresonance instruments (FT-MS) and matrix-assisted laserdesorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS).

Quantification of Bioenergetic Molecule Levels:

Environmental influencers (e.g., MIMs or Epi-shifters) may be identifiedby changes in cellular bioenergetic molecule levels (e.g., ATP,pyruvate, ADP, NADH, NAD, NADPH, NADP, acetylCoA, FADH2) of cells towhich a candidate epi-shifter has been applied. Exemplary assays ofbioenergetic molecule levels use colorometric, fluorescence, and/orbioluminescent-based methods. Examples of such assays are providedbelow.

Levels of ATP within cells can be measured with a number of assays andsystems known in the art. For example, in one system, cytoplasmic ATPreleased from lysed cells reacts with luciferin and the enzymeluciferase to produce light. This bioluminescence is measured by abioluminometer and the intracellular ATP concentration of the lysedcells can be calculated (EnzyLight™ ATP Assay Kit (EATP-100), BioAssaySystems, Hayward, Calif.). In another system, for example, both ATP andits dephosphorylated form, ADP, are calculated via bioluminescence;after ATP levels are calculated, ADP is transformed into ATP and thendetected and calculated using the same luciferase system (ApoSENSOR™ADP/ATP Ratio Assay Kit, BioVision Inc., Mountain View, Calif.).

Pyruvate is an important intermediate in cellular metabolic pathways.Pyruvate may be converted into carbohydrate via gluconeogenesis,converted into fatty acid or metabolized via acetyl CoA, or convertedinto alanine or ethanol, depending upon the metabolic state of a cell.Thus detection of pyruvate levels provides a measure of the metabolicactivity and state of a cell sample. One assay to detect pyruvate, forexample, uses both a colorimetric and fluorimetric to detect pyruvateconcentrations within different ranges (EnzyChrom™ Pyruvate Assay Kit(Cat#EPYR-100), BioAssay Systems, Hayward, Calif.).

Environmental influencers (e.g., MIMs or Epi-shifters) may influence theprocess of oxidative phosphorylation carried out by mitochondria incells, which are involved in the generation and maintenance ofbioenergetic molecules in cells. In addition to assays that detectchanges in cellular energetics in cell cultures and samples directly(described below), assays exist that detect and quantify the effects ofcompounds on discrete enzymes and complexes of mitochondria in cells.For example, the MT-OXC MitoTox™ Complete OXPHOS Activity Assay(MitoSciences Inc., Eugene, Oreg.) can detect and quantify the effectsof compounds applied directly to complexes I to V extracted frommitochondria. Assays for the detection and quantification of effects onindividual mitochondrial complexes such as NADH dehydrogenase (ComplexI), cytochrome c oxidase (Complex IV) and ATP synthase (Complex V) arealso available (MitoSciences Inc., Eugene, Oreg.).

Measurement of Cellular Energetics:

Environmental influencers (e.g., MIMs or Epi-shifters) may also beidentified by changes in cellular energetics. One example of themeasurement of cellular energetics are the real-time measures of theconsumption of molecular oxygen and/or the change in pH of the media ofa cell culture. For example, the ability of a potential epi-shifter tomodulate the metabolic state of a cell may be analyzed using, forexample, the XF24 Analyzer (Seahorse, Inc.). This technology allows forreal time detection of oxygen and pH changes in a monolayer of cells inorder to evaluate the bioenergetics of a cell microenvironment. The XF24Analyzer measures and compares the rates of oxygen consumption (OCR),which is a measure of aerobic metabolism, and extracellularacidification (ECAR), which is a measure of glycolysis, both keyindicators of cellular energetics.

Measurement of Oxidative Phosphorylation and Mitochondrial Function

Oxidative Phosphorylation is a process by which ATP is generated via theoxidation of nutrient compounds, carried out in eukaryotes via proteincomplexes embedded in the membranes of mitochondria. As the primarysource of ATP in the cells of most organisms, changes in oxidativephosphorylation activity can strongly alter metabolism and energybalance within a cell. In some embodiments of the invention,environmental influencers (e.g., MIMs or Epi-shifters) may be detectedand/or identified by their effects on oxidative phosphorylation. In someembodiments, environmental influencers (e.g., MIMs or Epi-shifters) maybe detected and/or identified by their effects on specific aspects ofoxidative phosphorylation, including, but not limited to, the electrontransport chain and ATP synthesis.

The membrane-embedded protein complexes of the mitochrondria that carryout processes involved in oxidative phosphorylation perform specifictasks and are numbered I, II, III and IV. These complexes, along withthe trans-inner membrane ATP synthase (also known as Complex V), are thekey entities involved in the oxidative phosphorylation process. Inaddition to assays that can examine the effects of environmentalinfluencers (e.g., MIMs or Epi-shifters) on mitochondrial function ingeneral and the oxidative phosphorylation process in particular, assaysare available that can be used to examine the effects of an epi-shifteron an individual complex separately from other complexes.

Complex I, also known as NADH-coenzyme Q oxidoreductase or NADHdehydrogenase, is the first protein in the electron transport chain. Insome embodiments, the detection and quantification of the effect of anepi-shifter on the production of NAD⁺ by Complex I may be performed. Forexample, the complex can be immunocaptured from a sample in a 96-wellplate; the oxidation of NADH to NAD⁺ takes place concurrently with thereduction of a dye molecule which has an increased absorbance at 450 nM(Complex I Enzyme Activity Microplate Assay Kit, MitoSciences Inc.,Eugene, Oreg.).

Complex IV, also known as cytochrome c oxidase (COX), is the lastprotein in the electron transport chain. In some embodiments, thedetection and quantification of the effect of an epi-shifter on theoxidation of cytochrome c and the reduction of oxygen to water byComplex IV may be performed. For example, COX can be immunocaptured in amicrowell plate and the oxidation of COX measured with a colorimetricassay (Complex IV Enzyme Activity Microplate Assay Kit, MitoSciencesInc., Eugene, Oreg.).

The final enzyme in the oxidative phosphorylation process is ATPsynthase (Complex V), which uses the proton gradient created by theother complexes to power the synthesis of ATP from ADP. In someembodiments, the detection and quantification of the effect of anepi-shifter on the activity of ATP synthase may be performed. Forexample, both the activity of ATP synthase and the amount of ATPsynthase in a sample may be measured for ATP synthase that has beenimmunocaptured in a microwell plate well. The enzyme can also functionas an ATPase under certain conditions, thus in this assay for ATPsynthase activity, the rate at which ATP is reduced to ADP is measuredby detecting the simultaneous oxidation of NADH to NAD⁺. The amount ofATP is calculated using a labeled antibody to ATPase (ATP synthaseDuplexing (Activity+Quantity) Microplate Assay Kit, MitoSciences Inc.,Eugene, Oreg.). Additional assays for oxidative phosphorylation includeassays that test for effects on the activity of Complexes II and III.For example, the MT-OXC MitoTox™ Complete OXPHOS System (MitoSciencesInc., Eugene, Oreg.) can be used to evaluate effects of a compound onComplex II and III as well as Complex I, IV and V, to provide data onthe effects of a compound on the entire oxidative phosphorylationsystem.

As noted above, real-time observation of intact cell samples can be madeusing probes for changes in oxygen consumption and pH in cell culturemedia. These assays of cell energetics provide a broad overview ofmitochondrial function and the effects of potential environmentalinfluencers (e.g., MIMs or Epi-shifters) on the activity of mitochondriawithin the cells of the sample.

Environmental influencers (e.g., MIMs or Epi-shifters) may also affectmitochondrial permeability transition (MPT), a phenomena in which themitochondrial membranes experience an increase in permeability due tothe formation of mitochondrial permeability transition pores (MPTP). Anincrease in mitochondrial permeability can lead to mitochondrialswelling, an inability to conduct oxidative phosphorylation and ATPgeneration and cell death. MPT may be involved with induction ofapoptosis. (See, for example, Halestrap, A. P., Biochem. Soc. Trans.34:232-237 (2006) and Lena, A. et al. Journal of Translational Med.7:13-26 (2009), hereby incorporated by reference in their entirety.)

In some embodiments, the detection and quantification of the effect ofan environmental influencer (e.g., MIM or epi-shifter) on the formation,discontinuation and/or effects of MPT and MPTPs are measured. Forexample, assays can detect MPT through the use of specialized dyemolecules (calcein) that are localized within the inner membranes ofmitochondria and other cytosolic compartments. The application ofanother molecule, CoCl₂, serves to squelch the fluorescence of thecalcein dye in the cytosol. CoCl₂ cannot access, however, the interiorof the mitochondria, thus the calcein fluorescence in the mitochondriais not squelched unless MPT has occurred and CoCl₂ can access theinterior of the mitochondra via MPTPs. Loss of mitochondrial-specificfluorescence signals that MPT has occurred. Flow cytometry can be usedto evaluate cellular and organelle fluorescence (MitoProbe™ TransitionPore Assay Kit, Molecular Probes, Eugene, Oreg.). Additional assaysutilize a fluorescence microscope for evaluating experimental results(Image-iT™ LIVE Mitochondrial Transition Pore Assay Kit, MolecularProbes, Eugene, Oreg.).

Measurement of Cellular Proliferation and Inflammation

In some embodiments of the invention, environmental influencers (e.g.,MIMs or Epi-shifters) may be identified and evaluated by their effectson the production or activity of molecules associated with cellularproliferation and/or inflammation. These molecules include, but are notlimited to, cytokines, growth factors, hormones, components of theextra-cellular matrix, chemokines, neuropeptides, neurotransmitters,neurotrophins and other molecules involved in cellular signaling, aswell as intracellular molecules, such as those involved in signaltransduction.

Vascular endothelial growth factor (VEGF) is a growth factor with potentangiogenic, vasculogenic and mitogenic properties. VEGF stimulatesendothelial permeability and swelling and VEGF activity is implicated innumerous diseases and disorders, including rheumatoid arthritis,metastatic cancer, age-related macular degeneration and diabeticretinopathy.

In some embodiments of the invention, an environmental influencer (e.g.,MIM or Epi-shifter) may be identified and characterized by its effectson the production of VEGF. For example, cells maintained in hypoxicconditions or in conditions mimicking acidosis will exhibit increasedVEGF production. VEGF secreted into media can be assayed using an ELISAor other antibody-based assays, using available anti-VEGF antibodies(R&D Systems, Minneapolis, Minn.). In some embodiments of the invention,an Epi-shifter may be identified and/or characterized based on itseffect(s) on the responsiveness of cells to VEGF and/or based on itseffect(s) on the expression or activity of the VEGF receptor.

Implicated in both healthy immune system function as well as inautoimmune diseases, tumor necrosis factor (TNF) is a key mediator ofinflammation and immune system activation. In some embodiments of theinvention, an Epi-shifter may be identified and characterized by itseffects on the production or the activity of TNF. For example, TNFproduced by cultured cells and secreted into media can be quantified viaELISA and other antibody-based assays known in the art. Furthermore, insome embodiments an environmental influencer may be identified andcharacterized by its effect(s) on the expression of receptors for TNF(Human TNF RI Duoset, R&D Systems, Minneapolis, Minn.).

The components of the extracellular matrix (ECM) play roles in both thestructure of cells and tissues and in signaling processes. For example,latent transforming growth factor beta binding proteins are ECMcomponents that create a reservoir of transforming growth factor beta(TGFβ) within the ECM. Matrix-bound TGFβ can be released later duringthe process of matrix remodeling and can exert growth factor effects onnearby cells (Dallas, S. Methods in Mol. Biol. 139:231-243 (2000)).

In some embodiments, an environmental influencer (e.g., MIM orEpi-shifter) may be identified or characterized by its effect(s) on thecreation of ECM by cultured cells. Researchers have developed techniqueswith which the creation of ECM by cells, as well as the composition ofthe ECM, can be studied and quantified. For example, the synthesis ofECM by cells can be evaluated by embedding the cells in a hydrogelbefore incubation. Biochemical and other analyses are performed on theECM generated by the cells after cell harvest and digestion of thehydrogel (Strehin, I. and Elisseeff, J. Methods in Mol. Bio. 522:349-362(2009)).

In some embodiments, the effect of environmental influencer (e.g., MIMor epi: shifter) on the production, status of or lack of ECM or one ofits components in an organism may be identified or characterized.Techniques for creating conditional knock-out (KO) mice have beendeveloped that allow for the knockout of particular ECM genes only indiscrete types of cells or at certain stages of development (Brancaccio,M. et al. Methods in Mol. Bio. 522:15-50 (2009)). The effect of theapplication or administration of an epi-shifter or potential epi-shifteron the activity or absence of a particular ECM component in a particulartissue or at a particular stage of development may thus be evaluated.

Measurement of Plasma Membrane Integrity and Cell Death

Environmental influencers (e.g., MIMs or Epi-shifters) may be identifiedby changes in the plasma membrane integrity of a cell sample and/or bychanges in the number or percentage of cells that undergo apoptosis,necrosis or cellular changes that demonstrate an increased or reducedlikelihood of cell death.

An assay for lactate dehydrogenase (LDH) can provide a measurement ofcellular status and damage levels. LDH is a stable and relativelyabundant cytoplasmic enzyme. When plasma membranes lose physicalintegrity, LDH escapes to the extracellular compartment. Higherconcentrations of LDH correlate with higher levels of plasma membranedamage and cell death. Examples of LDH assays include assays that use acolorimetric system to detect and quantify levels of LDH in a sample,wherein the reduced form of a tetrazolium salt is produced via theactivity of the LDH enzyme (QuantiChrom™ Lactate Dehydrogenase Kit(DLDH-100), BioAssay Systems, Hayward, Calif.; LDH CytotoxicityDetection Kit, Clontech, Mountain View, Calif.).

Apoptosis is a process of programmed cell death that may have a varietyof different initiating events. A number of assays can detect changes inthe rate and/or number of cells that undergo apoptosis. One type ofassay that is used to detect and quantify apoptosis is a capase assay.Capases are aspartic acid-specific cysteine proteases that are activatedvia proteolytic cleavage during apoptosis. Examples of assays thatdetect activated capases include PhiPhiLux® (OncoImmunin, Inc.,Gaithersburg, Md.) and Caspase-Glo® 3/7 Assay Systems (Promega Corp.,Madison, Wis.). Additional assays that can detect apoptosis and changesin the percentage or number of cells undergoing apoptosis in comparativesamples include TUNEL/DNA fragmentation assays. These assays detect the180 to 200 base pair DNA fragments generated by nucleases during theexecution phase of apoptosis. Exemplary TUNEL/DNA fragmentation assaysinclude the In Situ Cell Death Detection Kit (Roche Applied Science,Indianapolis, Ind.) and the DeadEnd™ Colorimetric and Fluorometric TUNELSystems (Promega Corp., Madison, Wis.).

Some apoptosis assays detect and quantify proteins associated with anapoptotic and/or a non-apoptotic state. For example, the MultiTox-FluorMultiplex Cytotoxicity Assay (Promega Corp., Madison, Wis.) uses asingle substrate, fluorimetric system to detect and quantify proteasesspecific to live and dead cells, thus providing a ratio of living cellsto cells that have undergone apoptosis in a cell or tissue sample.

Additional assays available for detecting and quantifying apoptosisinclude assays that detect cell permeability (e.g., APOPercentage™APOPTOSIS Assay, Biocolor, UK) and assays for Annexin V (e.g., AnnexinV-Biotin Apoptosis Detection Kit, BioVision Inc., Mountain View,Calif.).

IV. Treatment of Oncological Disorders

The present invention provides methods of treating or preventing anoncological disorder in a human, comprising administering anenvironmental influencer to the human in an amount sufficient to treator prevent the oncological disorder, thereby treating or preventing theoncological disorder. In one embodiment, the environmental influencer isnot CoQ10.

As used herein, “oncological disorder” refers to all types of cancer orneoplasm or malignant tumors found in humans, including, but not limitedto: leukemias, lymphomas, melanomas, carcinomas and sarcomas. As usedherein, the terms or language “oncological disorder”, “cancer,”“neoplasm,” and “tumor,” are used interchangeably and in either thesingular or plural form, refer to cells that have undergone a malignanttransformation that makes them pathological to the host organism.Primary cancer cells (that is, cells obtained from near the site ofmalignant transformation) can be readily distinguished fromnon-cancerous cells by well-established techniques, particularlyhistological examination. The definition of a cancer cell, as usedherein, includes not only a primary cancer cell, but also cancer stemcells, as well as cancer progenitor cells or any cell derived from acancer cell ancestor. This includes metastasized cancer cells, and invitro cultures and cell lines derived from cancer cells. When referringto a type of cancer that normally manifests as a solid tumor, a“clinically detectable” tumor is one that is detectable on the basis oftumor mass; e.g., by procedures such as CAT scan, MR imaging, X-ray,ultrasound or palpation, and/or which is detectable because of theexpression of one or more cancer-specific antigens in a sampleobtainable from a patient.

The term “sarcoma” generally refers to a tumor which is made up of asubstance like the embryonic connective tissue and is generally composedof closely packed cells embedded in a fibrillar or homogeneoussubstance. Examples of sarcomas which can be treated with anenvironmental influencer of the invention include, but are not limitedto, a chondrosarcoma, fibrosarcoma, lymphosarcoma, melanosarcoma,myxosarcoma, osteosarcoma, Abemethy's sarcoma, adipose sarcoma,liposarcoma, alveolar soft part sarcoma, ameloblastic sarcoma, botryoidsarcoma, chloroma sarcoma, chorio carcinoma, embryonal sarcoma, Wilms'tumor sarcoma, endometrial sarcoma, stromal sarcoma, Ewing's sarcoma,fascial sarcoma, fibroblastic sarcoma, giant cell sarcoma, granulocyticsarcoma, Hodgkin's sarcoma, idiopathic multiple pigmented hemorrhagicsarcoma, immunoblastic sarcoma of B cells, lymphoma, immunoblasticsarcoma of T-cells, Jensen's sarcoma, Kaposi's sarcoma, Kupffer cellsarcoma, angiosarcoma, leukosarcoma, malignant mesenchymoma sarcoma,parosteal sarcoma, reticulocytic sarcoma, Rous sarcoma, serocysticsarcoma, synovial sarcoma, and telangiectaltic sarcoma.

The term “melanoma” is taken to mean a tumor arising from themelanocytic system of the skin and other organs. Melanomas which can betreated with an environmental influencer of the invention include, butare not limited to, for example, acral-lentiginous melanoma, amelanoticmelanoma, benign juvenile melanoma, Cloudman's melanoma, S91 melanoma,Harding-Passey melanoma, juvenile melanoma, lentigo maligna melanoma,malignant melanoma, nodular melanoma, subungal melanoma, and superficialspreading melanoma.

The term “carcinoma” refers to a malignant new growth made up ofepithelial cells tending to infiltrate the surrounding tissues and giverise to metastases. Carcinomas which can be treated with anenvironmental influencer of the invention include, but are not limitedto, for example, acinar carcinoma, acinous carcinoma, adenocysticcarcinoma, adenoid cystic carcinoma, carcinoma adenomatosum, carcinomaof adrenal cortex, alveolar carcinoma, alveolar cell carcinoma, basalcell carcinoma, carcinoma basocellulare, basaloid carcinoma,basosquamous cell carcinoma, bronchioalveolar carcinoma, bronchiolarcarcinoma, bronchogenic carcinoma, cerebriform carcinoma,cholangiocellular carcinoma, chorionic carcinoma, colloid carcinoma,comedo carcinoma, corpus carcinoma, cribriform carcinoma, carcinoma encuirasse, carcinoma cutaneum, cylindrical carcinoma, cylindrical cellcarcinoma, duct carcinoma, carcinoma durum, embryonal carcinoma,encephaloid carcinoma, epiermoid carcinoma, carcinoma epithelialeadenoides, exophytic carcinoma, carcinoma ex ulcere, carcinoma fibrosum,gelatiniform carcinoma, gelatinous carcinoma, giant cell carcinoma,carcinoma gigantocellulare, glandular carcinoma, granulosa cellcarcinoma, hair-matrix carcinoma, hematoid carcinoma, hepatocellularcarcinoma, Hurthle cell carcinoma, hyaline carcinoma, hypemephroidcarcinoma, infantile embryonal carcinoma, carcinoma in situ,intraepidermal carcinoma, intraepithelial carcinoma, Krompecher'scarcinoma, Kulchitzky-cell carcinoma, large-cell carcinoma, lenticularcarcinoma, carcinoma lenticulare, lipomatous carcinoma, lymphoepithelialcarcinoma, carcinoma medullare, medullary carcinoma, melanoticcarcinoma, carcinoma molle, mucinous carcinoma, carcinoma muciparum,carcinoma mucocellulare, mucoepidermoid carcinoma, carcinoma mucosum,mucous carcinoma, carcinoma myxomatodes, nasopharyngeal carcinoma, oatcell carcinoma, carcinoma ossificans, osteoid carcinoma, papillarycarcinoma, periportal carcinoma, preinvasive carcinoma, prickle cellcarcinoma, pultaceous carcinoma, renal cell carcinoma of kidney, reservecell carcinoma, carcinoma sarcomatodes, schneiderian carcinoma,scirrhous carcinoma, carcinoma scroti, signet-ring cell carcinoma,carcinoma simplex, small-cell carcinoma, solanoid carcinoma, spheroidalcell carcinoma, spindle cell carcinoma, carcinoma spongiosum, squamouscarcinoma, squamous cell carcinoma, string carcinoma, carcinomatelangiectaticum, carcinoma telangiectodes, transitional cell carcinoma,carcinoma tuberosum, tuberous carcinoma, verrucous carcinoma, andcarcinoma villosum.

In general, an environmental influencer may be used to prophylacticallyor therapeutically treat any neoplasm. In one embodiment, theenvironmental influencers of the invention are used to treat solidtumors. In various embodiments of the invention, an environmentalinfluencer (e.g., CoQ10) is used for treatment, of various types of skincancer (e.g., Squamous cell Carcinoma or Basal Cell Carcinoma), livercancer, pancreatic cancer, breast cancer, prostate cancer, liver cancer,or bone cancer. In one embodiment, an environmental influencer, e.g.,CoQ10, is used for treatment of a skin oncological disorder including,but not limited to, squamous cell carcinomas (including SCCIS (in situ)and more aggressive squamous cell carcinomas), basal cell carcinomas(including superficial, nodular and infiltrating basal cell carcinomas),melanomas, and actinic keratosis. However, treatment using anenvironmental influencer is not limited to the foregoing types ofcancers. Examples of cancers amenable to treatment with an environmentalinfluencer include, but are not limited to, cancer of the brain, headand neck, prostate, breast, testicular, pancreas, liver, colon, bladder,kidney, lung, non-small cell lung, melanoma, mesothelioma, uterus,cervix, ovary, sarcoma, bone, stomach and Medulloblastoma.

Additional cancers which can be treated with an environmental influencerof the invention include, for example, Hodgkin's Disease, Non-Hodgkin'sLymphoma, multiple myeloma, neuroblastoma, breast cancer, ovariancancer, lung cancer, rhabdomyosarcoma, primary thrombocytosis, primarymacroglobulinemia, small-cell lung tumors, primary brain tumors, stomachcancer, colon cancer, malignant pancreatic insulanoma, malignantcarcinoid, urinary bladder cancer, premalignant skin lesions, testicularcancer, lymphomas, thyroid cancer, neuroblastoma; esophageal cancer,genitourinary tract cancer, malignant hypercalcemia, cervical cancer,endometrial cancer, adrenal cortical cancer, and prostate cancer. In oneembodiment, the oncological disorder or cancer which can be treated withthe environmental influencer, e.g., CoQ10, is not melanoma.

The present invention further provides methods of treating or preventingan oncological disorder in a human, comprising selecting a human subjectsuffering from an oncological disorder, and administering to said humana therapeutically effective amount of an Env-influencer capable ofblocking anaerobic use of glucose and augmenting mitochondrial oxidativephosphorylation, thereby treating or preventing the oncologicaldisorder.

The definition of a cancer cell, as used herein, is intended to includea cancer cell that produces energy by anaerobic glycolysis (e.g.,glycolysis followed by lactic acid fermantion in the cytosol), aerobicglycolysis or mitochondrial oxidative phosphorylation (e.g., glycolysisfollowed by oxidation of pyruvate in the mitochondria), or a combinationof anaerobic glycolysis and aerobic glycolysis. In one embodiment, acancer cell produces energy predominantly by anaerobic glycolysis (e.g.,at least 50%, 60%, 70%, 80%, 90%, 95% or more of the cell's energy isproduced by anaerobic glycolysis). In one embodiment, a cancer cellproduces energy predominantly by aerobic glycolysis (e.g., at least 50%,60%, 70%, 80%, 90%, 95% or more of the cell's energy is produced byanaerobic glycolysis). The definition of cancer cells, as used herein,is also intended to include a cancer cell population or mixture ofcancer cells comprising cells that produce energy by anaerobicglycolysis and cells that produce energy by aerobic glycolysis. In oneembodiment, a cancer cell population comprises predominantly cells thatproduce energy by anaerobic glycolysis (e.g., at least 50%, 60%, 70%,80%, 90%, 95% or more of the cells in the population produce energy byanaerobic glycolysis). In one embodiment, a cancer cell populationcomprises predominantly cells that produce energy by aerobic glycolysis(e.g., at least 50%, 60%, 70%, 80%, 90%, 95% or more of the cells in thepopulation).

As used herein, the phrase “anaerobic use of glucose” or “anaerobicglycolysis” or “glycolysis pathway” refers to cellular production ofenergy by glycolysis followed by lactic acid fermentation in thecytosol. For example, many cancer cells produce energy by anaerobicglycolysis.

As used herein, the phrase “aerobic glycolysis” or “mitochondrialoxidative phosphorylation” refers to cellular production of energy byglycolysis followed by oxidation of pyruvate in mitochondria.

As used herein, the phrase “capable of blocking anaerobic use of glucoseand augmenting mitochondrial oxidative phosphorylation” or “a shift fromglycolysis pathway to mitocondrial oxidative phosphorylation” refers tothe ability of an environmental influencer (e.g., an epitmetabolicshifter) to induce a shift or change in the metabolic state of a cellfrom anaerobic glycolysis to aerobic glycolysis or mitochondrialoxidative phosphorylation. As used herein, “shift (from glycolysis tomitochondrial oxidative phosphorylation)” refers to a reduction ofenergy dependency on the glycolysis pathway, preferably towards a levelseen in normal cells. Concommittantly, the absolute level ofmitocondrial oxidative phosphorylation may also reduce or decrease, butis preferably associated with increased efficiency of the mitocondrialoxidative phosphorylation.

In some embodiments of the invention, the oncological disorder beingtreated is not a disorder typically treated via topical administrationwith the expectation of systemic delivery of an active agent attherapeutically effective levels. As used herein, the phrase “not adisorder typically treated via topical administration” refers tooncological disorders that are not typically or routinely treated with atherapeutic agent via topical administrationbut rather are typicallytreated with a therapeutic agent via, for example, intravenousadministration. Oncological disorders not typically treated via topicaladministration include, but are not limited to, breast cancer, prostatecancer, liver cancer, pancreatic cancer, and bone cancer.

The present invention also provides a method for treating or preventingan aggressive oncological disorder in a human, comprising administeringan environmental infuencer to the human at a selected lower dose thanthe dosage regimen used or selected for less aggressive ornon-aggressive oncological disorders, thereby treating or preventing theaggressive oncological disorder. In a related aspect, the inventionprovides a method for treating or preventing a non-aggressiveoncological disorder in a human, comprising administering anenvironmental influencer to the human at a selected higher dose over thedosage regimen used or selected for aggressive oncological disorders,thereby treating or preventing the non-aggressive oncological disorder.

As used herein, the term “aggressive oncological disorder” refers to anoncological disorder involving a fast-growing tumor. An aggressiveoncological disorder typically does not respond or responds poorly totherapeutic treatment. Examples of an aggressive oncological disorderinclude, but are not limited to, pancreatic carcinoma, hepatocellularcarcinoma, Ewing's sarcoma, metastatic breast cancer, metastaticmelanoma, brain cancer (astrocytoma, glioblastoma), neuroendocrinecancer, colon cancer, lung cancer, osteosarcoma, androgen-independentprostate cancer, ovarian cancer and non-Hodgkin's Lymphoma.

As used herein, the term “non-aggressive oncological disorder” refers toan oncological disorder involving a slow-growing tumor. A non-aggressiveoncological disorder typically responds favorably or moderately totherapeutic treatment. Examples of a non-aggressive oncological disorderinclude, but are not limited to, non-metastatic breast cancer,androgen-dependent prostate cancer, small cell lung cancer and acutelymphocytic leukemia. In one embodiment, non-aggressive oncologicaldisorders include any oncological disorder that is not an aggressiveoncological disorder.

A selected lower dosage of CoQ10 for the treatment of aggressiveoncological disorders is intended to include a dosage that is lower thana dosage regimen that is typically used or selected for less aggressiveor non-aggressive oncological disorders. In various embodiments, theselected lower dosage of CoQ10 is about 1.5-fold lower, about 2 foldlower, about 3-fold lower, about 4-fold lower, about 5-fold lower orabout 10-fold lower than a dosage regimen that is typically used orselected for less aggressive or non-aggressive oncological disorders. Itwill be understood that a selected lower dosage of CoQ10 also includes ashorter treatment time (e.g., 1.5 fold, 2 fold, 3 fold, 4 fold, 5 foldor 10 fold shorter treatment time) of CoQ10 or less frequentadministration (e.g., half as frequent, 3 fold, 4 fold, 5 fold, 10 fold,20 fold or 24 fold less frequent) of CoQ10 as compared to the treatmenttime or administration protocol typically used or selected for lessaggressive or on-aggressive oncological disorders. In variousembodiments, the selected lower dosage of coenzyme Q10 for the treatmentof aggressive oncological disorders includes about 0.0001 to about 5.0,about 0.001 to about 1.0, about 0.001 to about 0.5, about 0.001 to about0.4, about 0.001 to about 0.30, about 0.001 to about 0.25, about 0.001to 0.20, about 0.001 to about 0.12, or about 0.001 to about 0.09 mgCoQ10 per square centimeter of skin. In other embodiments, Coenzyme Q10is applied to the target tissue at a dose of about 0.0001, 0.001, 0.01,0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13,0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25,0.26, 0.27, 0.28, 0.29, 0.30, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37,0.38, 0.39, 0.40, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49or 0.5 mg CoQ10 per square centimeter of skin. It should be understoodthat ranges having any one of these values as the upper or lower limitsare also intended to be part of this invention, e.g., about 0.005 toabout 0.09 mg CoQ10 per square centimeter of skin.

A selected higher dosage of CoQ10 for the treatment of non-aggressiveoncological disorders is intended to include a dosage that is higherthan a dosage regimen that is typically used or selected for aggressiveoncological disorders. In various embodiments, the selected higherdosage of CoQ10 is about 1.5-fold, about 2 fold, about 3-fold, about4-fold, about 5-fold or about 10-fold higher than a dosage regimen thatis typically used or selected for aggressive oncological disorders. Itwill be understood that a selected lower dosage of CoQ10 also includes alonger treatment time (e.g., 1.5 fold, 2 fold, 3 fold, 4 fold, 5 fold or10 fold longer treatment time) of CoQ10 or more frequent administration(e.g., 1.5 fold, 2 fold, 3 fold, 4 fold, 5 fold, 10 fold, 20 fold or 24fold more frequent) of CoQ10 as compared to the treatment time oradministration protocol typically used or selected for aggressiveoncological disorders. In various embodiments, the selected higherdosage of coenzyme Q10 for the treatment of aggressive oncologicaldisorders includes about 0.001 to about 10.0, about 0.005 to about 10.0,about 0.01 to about 10.0, about 0.05 to about 5.0, about 0.05 to about2.0, about 0.05 to about 1.0, about 0.05 to about 0.7, about 0.10 toabout 0.50, or about 0.12 to 0.5 mg CoQ10 per square centimeter of skinIn other embodiments, Coenzyme Q10 is applied to the target tissue at adose of about 0.001, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08,0.09; 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20,0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.30, 0.31, 0.32,0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.40, 0.41, 0.42, 0.43, 0.44,0.45, 0.46, 0.47, 0.48, 0.49, 0.5 mg, 0.6 mg, 0.7 mg., 0.8 mg., 0.9 mgor 1.0 mg CoQ10 per square centimeter of skin. It should be understoodthat ranges having any one of these values as the upper or lower limitsare also intended to be part of this invention, e.g., about 0.15 toabout 0.5 mg CoQ10 per square centimeter of skin.

In one embodiment, an environmental influencer of the invention reducestumor size, inhibits tumor growth and/or prolongs the survival time of atumor-bearing subject. Accordingly, this invention also relates to amethod of treating tumors in a human or other animal by administering tosuch human or animal an effective, non-toxic amount of an environmentalinfluencer. One skilled in the art would be able, by routineexperimentation, to determine what an effective, non-toxic amount of anenvironmental influencer would be for the purpose of treatingmalignancies. For example, a therapeutically active amount of anenvironmental influencer may vary according to factors such as thedisease stage (e.g., stage I versus stage IV), age, sex, medicalcomplications (e.g., immunosuppressed conditions or diseases) and weightof the subject, and the ability of the environmental influencer toelicit a desired response in the subject. The dosage regimen may beadjusted to provide the optimum therapeutic response. For example,several divided doses may be administered daily, or the dose may beproportionally reduced as indicated by the exigencies of the therapeuticsituation.

V. Therapeutic Targets for Oncological Disorders

The present invention provides methods for identifying therapeutictargets for oncological disorders. The invention further providestherapeutic targets identified by such methods. The identification of atherapeutic target involves, generally, the exogenous application of anEnv-influencer or candidate Env-influencer to a cell or panel of celllines, and the subsequent evaluation of changes induced to a treatedcell as compared to a control, untreated cell. Induced cellular changeswhich are monitored include, but are not limited to, changes to themorphology, physiology or composition, e.g., RNA, protein, lipid ormetabolite levels, of the cell. Induced cellular changes as a result oftreatment by a candidate Env-influencer can be monitored by using any ofthe assays described herein. For example, changes in gene expression atthe mRNA level can be evaluated by real-time PCR arrays, while changesin gene expression at the protein level can be monitored by usingantibody microarrays and 2-D gel electrophoresis. Genes identified asbeing modulated by the candidate Env-influencer (e.g., at the mRNAand/or protein level) are then evaluated from a Systems Biologyperspective using pathway analysis (Ingenuity IPA software) and by areview of the known literature. Genes identified as potentialtherapeutic targets are next submitted to confirmatory assays such asWestern blot analysis, siRNA knock-down, or recombinant proteinproduction and characterization methods. Screening assays can then beused to identify modulators of the targets. Modulators of thetherapeutic targets are useful as novel therapeutic agents foroncological disorders. Modulators of therapeutic targets can beroutinely identified using screening assays described in detail herein,or by using routine methodologies known to the skilled artisan.

Genes identified herein as being modulated (e.g., upmodulated ordownmodulated, at either the mRNA or protein level) by theMIM/Epi-shifter, CoQ10, are drug targets of the invention. Drug targetsof the invention include, but are not limited to, the genes subsequentlylisted in Tables 1-28 herein. Based on the results of experimentsdescribed by Applicants herein, the key proteins modulated by Q10 areassociated with or can be classified into different pathways or groupsof molecules, including transcription factors, apoptotic response,pentose phosphate pathway, biosynthetic pathway, oxidative stress(pro-oxidant), membrane alterations, and oxidative phosphorylationmetabolism. The key proteins modulated by CoQ10, based on the resultsprovided herein, are summarized as follows. A key protein modulated byCoQ10 and which is a transcription factor is HNF4alpha. Key proteinsthat are modulated by CoQ10 and associated with the apoptotic responseinclude Bcl-xl, Bcl-xl, Bcl-xS, BNIP-2, Bcl-2, Birc6, Bcl-2-L11 (Bim),XIAP, BRAF, Bax, c-Jun, Bmf, PUMA, and cMyc. A key protein that ismodulated by CoQ10 and associated with the pentose phosphate pathway istransaldolase 1. Key proteins that are modulated by CoQ10 and associatedwith a biosynthetic pathway include COQ1, COQ3, COQ6, prenyltransferaseand 4-hydroxybenzoate. Key proteins that are modulated by CoQ10 andassociated with oxidative stress (pro-oxidant) include Neutrophilcytosolic factor 2, nitric oxide synthase 2A and superoxide dismutase 2(mitochondrial). Key proteins that are modulated by CoQ10 and associatedwith oxidative phosphorylation metabolism include Cytochrome c, complexI, complex II, complex III and complex IV. Further key proteins that aredirectly or indirectly modulated by CoQ10 include Foxo 3a, DJ-1, IDH-1,Cpt1C and Cam Kinase II.

Accordingly, in one embodiment of the invention, a drug target mayinclude HNF4-alpha, Bcl-xl, Bcl-xS, BNIP-2, Bcl-2, Birc6, Bcl-2-L11(Bim), XIAP, BRAF, Bax, c-Jun, Bmf, PUMA, cMyc, transaldolase 1, COQ1,COQ3, COQ6, prenyltransferase, 4-hydrobenzoate, neutrophil cytosolicfactor 2, nitric oxide synthase 2A, superoxide dismutase 2, VDAC, Baxchannel, ANT, Cytochrome c, complex 1, complex II, complex III, complexIV, Foxo 3a, DJ-1, IDH-1, Cpt1C and Cam Kinase II. In a preferredembodiment, a drug target may include HNF4A, Transaldolase, NM23 andBSCv. In one embodiment, the drug target is TNF4A. In one embodiment,the drug target is transaldolase. In one embodiment, the drug target isNM23. In one embodiment, the drug target is BSCv. Screening assaysuseful for identifying modulators of identified drug targets aredescribed below.

VI. Screening Assays

The invention also provides methods (also referred to herein as“screening assays”) for identifying modulators, i.e., candidate or testcompounds or agents (e.g., proteins, peptides, peptidomimetics,peptoids, small molecules or other drugs), which modulate the expressionand/or activity of an identified therapeutic target of the invention.Such assays typically comprise a reaction between a therapeutic targetof the invention and one or more assay components. The other componentsmay be either the test compound itself, or a combination of testcompounds and a natural binding partner of a marker of the invention.Compounds identified via assays such as those described herein may beuseful, for example, for treating or preventing a oncological disorder.

The test compounds used in the screening assays of the present inventionmay be obtained from any available source, including systematiclibraries of natural and/or synthetic compounds. Test compounds may alsobe obtained by any of the numerous approaches in combinatorial librarymethods known in the art, including: biological libraries; peptoidlibraries (libraries of molecules having the functionalities ofpeptides, but with a novel, non-peptide backbone which are resistant toenzymatic degradation but which nevertheless remain bioactive; see,e.g., Zuckermann et al., 1994, J. Med. Chem. 37:2678-85); spatiallyaddressable parallel solid phase or solution phase libraries; syntheticlibrary methods requiring deconvolution; the ‘one-bead one-compound’library method; and synthetic library methods using affinitychromatography selection. The biological library and peptoid libraryapproaches are limited to peptide libraries, while the other fourapproaches are applicable to peptide, non-peptide oligomer or smallmolecule libraries of compounds (Lam, 1997, Anticancer Drug Des.12:145).

Examples of methods for the synthesis of molecular libraries can befound in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad.Sci. U.S.A. 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA91:11422; Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho et al.(1993) Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed.Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061;and in Gallop et al. (1994) J. Med. Chem. 37:1233.

Libraries of compounds may be presented in solution (e.g., Houghten,1992, Biotechniques 13:412-421), or on beads (Lam, 1991, Nature354:82-84), chips (Fodor, 1993, Nature 364:555-556), bacteria and/orspores, (Ladner, U.S. Pat. No. 5,223,409), plasmids (Cull et al, 1992,Proc Natl Acad Sci USA 89:1865-1869) or on phage (Scott and Smith, 1990,Science 249:386-390; Devlin, 1990, Science 249:404-406; Cwirla et al,1990, Proc. Natl. Acad. Sci. 87:6378-6382; Felici, 1991, J. Mol. Biol.222:301-310; Ladner, supra.).

The screening methods of the invention comprise contacting a cell with atest compound and determining the ability of the test compound tomodulate the expression and/or activity of a therapeutic target of theinvention in the cell. The expression and/or activity of a therapeutictarget of the invention can be determined as described herein. Theexpression and/or activity of a therapeutic target of the invention canalso be determined by using routine methods known to the skilledartisan. In one embodiment, a compound is selected based on its abilityto increase expression and/or activity of a therapeutic target of theinvention. In one embodiment, a compound is selected based on itsability increase expression and/or activity of a therapeutic targetselected from the protein listed in Tables 1-28, wherein the therapeutictarget is upmodulated by CoQ10 (e.g., exhibits a positive-fold change).In one embodiment, a compound is selected based on its ability todecrease expression and/or activity of a therapeutic target of theinvention. In one embodiment, a compound is selected based on itsability to decrease expression and/or activity of a therapeutic targetselected from the proteins listed in Tables 1-28, wherein thetherapeutic targetis is downmodulated by CoQ10 (e.g., exhibits anegative-fold change).

In another embodiment, the invention provides assays for screeningcandidate or test compounds which are substrates of a therapeutic targetof the invention or biologically active portions thereof. In yet anotherembodiment, the invention provides assays for screening candidate ortest compounds which bind to a therapeutic target of the invention orbiologically active portions thereof. Determining the ability of thetest compound to directly bind to a therapeutic target can beaccomplished, for example, by coupling the compound with a radioisotopeor enzymatic label such that binding of the compound to the drug targetcan be determined by detecting the labeled marker compound in a complex.For example, compounds (e.g., marker substrates) can be labeled with¹³¹I, ¹²⁵I, ³⁵S, ¹⁴C, or ³H, either directly or indirectly, and theradioisotope detected by direct counting of radioemission or byscintillation counting. Alternatively, assay components can beenzymatically labeled with, for example, horseradish peroxidase,alkaline phosphatase, or luciferase, and the enzymatic label detected bydetermination of conversion of an appropriate substrate to product.

This invention further pertains to novel agents identified by theabove-described screening assays. Accordingly, it is within the scope ofthis invention to further use an agent identified as described herein inan appropriate animal model. For example, an agent capable of modulatingthe expression and/or activity of a marker of the invention identifiedas described herein can be used in an animal model to determine theefficacy, toxicity, or side effects of treatment with such an agent.Alternatively, an agent identified as described herein can be used in ananimal model to determine the mechanism of action of such an agent.Furthermore, this invention pertains to uses of novel agents identifiedby the above-described screening assays for treatment as describedabove.

VII. Pharmaceutical Compositions and Pharmaceutical Administration

The environmental influencers of the invention can be incorporated intopharmaceutical compositions suitable for administration to a subject.Typically, the pharmaceutical composition comprises an environmentalinfluencer of the invention and a pharmaceutically acceptable carrier.As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like that arephysiologically compatible. Examples of pharmaceutically acceptablecarriers include one or more of water, saline, phosphate bufferedsaline, dextrose, glycerol, ethanol and the like, as well ascombinations thereof. In many cases, it will be preferable to includeisotonic agents, for example, sugars, polyalcohols such as mannitol,sorbitol, or sodium chloride in the composition. Pharmaceuticallyacceptable carriers may further include minor amounts of auxiliarysubstances such as wetting or emulsifying agents, preservatives orbuffers, which enhance the shelf life or effectiveness of theenvironmental influencer.

The compositions of this invention may be in a variety of forms. Theseinclude, for example, liquid, semi-solid and solid dosage forms, such asliquid solutions (e.g., injectable and infusible solutions), dispersionsor suspensions, tablets, pills, powders, creams, lotions, liniments,ointments or pastes, drops for administration to the eye, ear or nose,liposomes and suppositories. The preferred form depends on the intendedmode of administration and therapeutic application.

The environmental influencers of the present invention can beadministered by a variety of methods known in the art. For manytherapeutic applications, the preferred route/mode of administration issubcutaneous injection, intravenous injection or infusion. As will beappreciated by the skilled artisan, the route and/or mode ofadministration will vary depending upon the desired results. In certainembodiments, the active compound may be prepared with a carrier thatwill protect the compound against rapid release, such as a controlledrelease formulation, including implants, transdermal patches, andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Manymethods for the preparation of such formulations are patented orgenerally known to those skilled in the art. See, e.g., Sustained andControlled Release Drug Delivery Systems, J. R. Robinson, ed., MarcelDekker, Inc., New York, 1978. In one embodiment, the mode ofadministration is parenteral (e.g., intravenous, subcutaneous,intraperitoneal, intramuscular). In one embodiment, the environmentalinfluencer is administered by intravenous infusion or injection. Inanother embodiment, the environmental influencer is administered byintramuscular or subcutaneous injection. In a preferred embodiment, theenvironmental influencer is administered topically.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, dispersion, liposome, or other orderedstructure suitable to high drug concentration. Sterile injectablesolutions can be prepared by incorporating the active compound (i.e.,environmental influencer) in the required amount in an appropriatesolvent with one or a combination of ingredients enumerated above, asrequired, followed by filtered sterilization. Generally, dispersions areprepared by incorporating the active compound into a sterile vehiclethat contains a basic dispersion medium and the required otheringredients from those enumerated above. In the case of sterile,lyophilized powders for the preparation of sterile injectable solutions,the preferred methods of preparation are vacuum drying and spray-dryingthat yields a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered solution thereof.The proper fluidity of a solution can be maintained, for example, by theuse of a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prolonged absorption of injectable compositions can be brought about byincluding in the composition an agent that delays absorption, forexample, monostearate salts and gelatin.

Techniques and formulations generally may be found in Remmington'sPharmaceutical Sciences, Meade Publishing Co., Easton, Pa. For systemicadministration, injection is preferred, including intramuscular,intravenous, intraperitoneal, and subcutaneous. For injection, thecompounds of the invention can be formulated in liquid solutions,preferably in physiologically compatible buffers such as Hank's solutionor Ringer's solution. In addition, the compounds may be formulated insolid form and redissolved or suspended immediately prior to use.Lyophilized forms are also included.

For oral administration, the pharmaceutical compositions may take theform of, for example, tablets or capsules prepared by conventional meanswith pharmaceutically acceptable excipients such as binding agents(e.g., pregelatinised maize starch, polyvinylpyrrolidone orhydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystallinecellulose or calcium hydrogen phosphate); lubricants (e.g., magnesiumstearate, talc or silica); disintegrants (e.g., potato starch or sodiumstarch glycolate); or wetting agents (e.g., sodium lauryl sulphate). Thetablets may be coated by methods well known in the art. Liquidpreparations for oral administration may take the form of, for example,solutions, syrups or suspensions, or they may be presented as a dryproduct for constitution with water or other suitable vehicle beforeuse. Such liquid preparations may be prepared by conventional means withpharmaceutically acceptable additives such as suspending agents (e.g.,sorbitol syrup, cellulose derivatives or hydrogenated edible fats);emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles(e.g., ationd oil, oily esters, ethyl alcohol or fractionated vegetableoils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates orsorbic acid). The preparations may also contain buffer salts, flavoring,coloring and sweetening agents as appropriate.

Preparations for oral administration may be suitably formulated to givecontrolled release of the active compound. For buccal administration thecompositions may take the form of tablets or lozenges formulated inconventional manner. For administration by inhalation, the compounds foruse according to the present invention are conveniently delivered in theform of an aerosol spray presentation from pressurized packs or anebuliser, with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof e.g., gelatin for use in an inhaler or insufflator may be formulatedcontaining a powder mix of the compound and a suitable powder base suchas lactose or starch.

The compounds may be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection may be presented in unit dosage form, e.g., in ampoules orin multi-dose containers, with an added preservative. The compositionsmay take such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient may be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

The compounds may also be formulated in rectal compositions such assuppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds mayalso be formulated as a depot preparation. Such long acting formulationsmay be administered by implantation (for example subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, thecompounds may be formulated with suitable polymeric or hydrophobicmaterials (for example as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration bile salts and fusidic acidderivatives in addition, detergents may be used to facilitatepermeation. Transmucosal administration may be through nasal sprays orusing suppositories. For topical administration, the compound(s) of theinvention are formulated into ointments, salves, gels, or creams asgenerally known in the art. A wash solution can be used locally to treatan injury or inflammation to accelerate healing.

The compositions may, if desired, be presented in a pack or dispenserdevice which may contain one or more unit dosage forms containing theactive ingredient. The pack may for example comprise metal or plasticfoil, such as a blister pack. The pack or dispenser device may beaccompanied by instructions for administration.

For therapies involving the administration of nucleic acids, thecompound(s) of the invention can be formulated for a variety of modes ofadministration, including systemic and topical or localizedadministration. Techniques and formulations generally may be found inRemmington's Pharmaceutical Sciences, Meade Publishing Co., Easton, Pa.For systemic administration, injection is preferred, includingintramuscular, intravenous, intraperitoneal, intranodal, andsubcutaneous. For injection, the compound(s) of the invention can beformulated in liquid solutions, preferably in physiologically compatiblebuffers such as Hank's solution or Ringer's solution. In addition, thecompound(s) may be formulated in solid form and redissolved or suspendedimmediately prior to use. Lyophilized forms are also included.

In one embodiment, the compositions comprising an Environmentalinfluencer are administered topically. It is preferable to present theactive ingredient, i.e. Env-influencer, as a pharmaceutical formulation.The active ingredient may comprise, for topical administration, fromabout 0.001% to about 20% w/w, by weight of the formulation in the finalproduct, although it may comprise as much as 30% w/w, preferably fromabout 1% to about 20% w/w of the formulation. The topical formulationsof the present invention, comprise an active ingredient together withone or more acceptable carrier(s) therefor and optionally any othertherapeutic ingredients(s). The carrier(s) should be “acceptable” in thesense of being compatible with the other ingredients of the formulationand not deleterious to the recipient thereof.

In treating a patient exhibiting a disorder of interest, atherapeutically effective amount of an agent or agents such as these isadministered. A therapeutically effective dose refers to that amount ofthe compound that results in amelioration of symptoms or a prolongationof survival in a patient.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD₅₀ (the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀.Compounds which exhibit large therapeutic indices are preferred. Thedata obtained from these cell culture assays and animal studies can beused in formulating a range of dosage for use in human. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized.

For any compound used in the method of the invention, thetherapeutically effective dose can be estimated initially from cellculture assays. For example, a dose can be formulated in animal modelsto achieve a circulating plasma concentration range that includes theIC₅₀ as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by HPLC.

The exact formulation, route of administration and dosage can be chosenby the individual physician in view of the patient's condition. (Seee.g. Fingl et al., in The Pharmacological Basis of Therapeutics, 1975,Ch. 1 p. 1). It should be noted that the attending physician would knowhow to and when to terminate, interrupt, or adjust administration due totoxicity, or to organ dysfunctions. Conversely, the attending physicianwould also know to adjust treatment to higher levels if the clinicalresponse were not adequate (precluding toxicity). The magnitude of anadministrated dose in the management of the oneogenic disorder ofinterest will vary with the severity of the condition to be treated andto the route of administration. The severity of the condition may, forexample, be evaluated, in part, by standard prognostic evaluationmethods. Further, the dose and perhaps dose frequency, will also varyaccording to the age, body weight, and response of the individualpatient. A program comparable to that discussed above may be used inveterinary medicine.

Depending on the specific conditions being treated, such agents may beformulated and administered systemically or locally. Techniques forformulation and administration may be found in Remington'sPharmaceutical Sciences, 18^(th) ed., Mack Publishing Co., Easton, Pa.(1990). Suitable routes may include oral, rectal, transdermal, vaginal,transmucosal, or intestinal administration; parenteral delivery,including intramuscular, subcutaneous, intramedullary injections, aswell as intrathecal, direct intraventricular, intravenous,intraperitoneal, intranasal, or intraocular injections, just to name afew.

The compositions described above may be administered to a subject in anysuitable formulation. In addition to treatment of a oncological disorderwith topical formulations of CoQ10, in other aspects of the inventionCoQ10 might be delivered by other methods. For example, CoQ10 might beformulated for parenteral delivery, e.g., for subcutaneous, intravenous,intramuscular, or intratumoral injection. Other methods of delivery, forexample, liposomal delivery or diffusion from a device impregnated withthe composition might be used. The compositions may be administered in asingle bolus, multiple injections, or by continuous infusion (forexample, intravenously or by peritoneal dialysis). For parenteraladministration, the compositions are preferably formulated in asterilized pyrogen-free form. Compositions of the invention can also beadministered in vitro to a cell (for example, to induce apoptosis in acancer cell in an in vitro culture) by simply adding the composition tothe fluid in which the cell is contained.

Depending on the specific conditions being treated, such agents may beformulated and administered systemically or locally. Techniques forformulation and administration may be found in Remington'sPharmaceutical Sciences, 18.^(th) ed., Mack Publishing Co., Easton, Pa.(1990). Suitable routes may include oral, rectal, transdermal, vaginal,transmucosal, or intestinal administration; parenteral delivery,including intramuscular, subcutaneous, intramedullary injections, aswell as intrathecal, direct intraventricular, intravenous,intraperitoneal, intranasal, or intraocular injections, just to name afew.

For injection, the agents of the invention may be formulated in aqueoussolutions, preferably in physiologically compatible buffers such asHanks's solution, Ringer's solution, or physiological saline buffer. Forsuch transmucosal administration, penetrants appropriate to the barrierto be permeated are used in the formulation. Such penetrants aregenerally known in the art.

Use of pharmaceutically acceptable carriers to formulate the compoundsherein disclosed for the practice of the invention into dosages suitablefor systemic administration is within the scope of the invention. Withproper choice of carrier and suitable manufacturing practice, thecompositions of the present invention, in particular, those formulatedas solutions, may be administered parenterally, such as by intravenousinjection. The compounds can be formulated readily usingpharmaceutically acceptable carriers well known in the art into dosagessuitable for oral administration. Such carriers enable the compounds ofthe invention to be formulated as tablets, pills, capsules, liquids,gels, syrups, slurries, suspensions and the like, for oral ingestion bya patient to be treated.

Agents intended to be administered intracellularly may be administeredusing techniques well known to those of ordinary skill in the art. Forexample, such agents may be encapsulated into liposomes, thenadministered as described above. Liposomes are spherical lipid bilayerswith aqueous interiors. All molecules present in an aqueous solution atthe time of liposome formation are incorporated into the aqueousinterior. The liposomal contents are both protected from the externalmicroenvironment and, because liposomes fuse with cell membranes, areefficiently delivered into the cell cytoplasm. Additionally, due totheir hydrophobicity, small organic molecules may be directlyadministered intracellularly.

Pharmaceutical compositions suitable for use in the present inventioninclude compositions wherein the active ingredients are contained in aneffective amount to achieve its intended purpose. Determination of theeffective amounts is well within the capability of those skilled in theart, especially in light of the detailed disclosure provided herein. Inaddition to the active ingredients, these pharmaceutical compositionsmay contain suitable pharmaceutically acceptable carriers comprisingexcipients and auxiliaries which facilitate processing of the activecompounds into preparations which can be used pharmaceutically. Thepreparations formulated for oral administration may be in the form oftablets, dragees, capsules, or solutions. The pharmaceuticalcompositions of the present invention may be manufactured in a mannerthat is itself known, e.g., by means of conventional mixing, dissolving,granulating, dragee-making, levitating, emulsifying, encapsulating,entrapping or lyophilizing processes.

Formulations suitable for topical administration include liquid orsemi-liquid preparations suitable for penetration through the skin tothe site of where treatment is required, such as liniments, lotions,creams, ointments or pastes, and drops suitable for administration tothe eye, ear, or nose. Drops according to the present invention maycomprise sterile aqueous or oily solutions or suspensions and may beprepared by dissolving the active ingredient in a suitable aqueoussolution of a bactericidal and/or fungicidal agent and/or any othersuitable preservative, and preferably including a surface active agent.The resulting solution may then be clarified and sterilized byfiltration and transferred to the container by an aseptic technique.Examples of bactericidal and fungicidal agents suitable for inclusion inthe drops are phenylmercuric nitrate or acetate (0.002%), benzalkoniumchloride (0.01%) and chlorhexidine acetate (0.01%). Suitable solventsfor the preparation of an oily solution include glycerol, dilutedalcohol and propylene glycol.

Lotions according to the present invention include those suitable forapplication to the skin or eye. An eye lotion may comprise a sterileaqueous solution optionally containing a bactericide and may be preparedby methods similar to those for the preparation of drops. Lotions orliniments for application to the skin may also include an agent tohasten drying and to cool the skin, such as an alcohol or acetone,and/or a moisturizer such as glycerol or an oil such as castor oil orarachis oil.

Creams, ointments or pastes according to the present invention aresemi-solid formulations of the active ingredient for externalapplication. They may be made by mixing the active ingredient infinely-divided or powdered form, alone or in solution or suspension inan aqueous or non-aqueous fluid, with the aid of suitable machinery,with a greasy or non-greasy basis. The basis may comprise hydrocarbonssuch as hard, soft or liquid paraffin, glycerol, beeswax, a metallicsoap; a mucilage; an oil of natural origin such as almond, corn,arachis, castor or olive oil; wool fat or its derivatives, or a fattyacid such as stearic or oleic acid together with an alcohol such aspropylene glycol or macrogels. The formulation may incorporate anysuitable surface active agent such as an anionic, cationic or non-ionicsurface active such as sorbitan esters or polyoxyethylene derivativesthereof. Suspending agents such as natural gums, cellulose derivativesor inorganic materials such as silicaceous silicas, and otheringredients such as lanolin, may also be included.

Pharmaceutical formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble form.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilizers or agents which increase the solubility of thecompounds to allow for the preparation of highly concentrated solutions.

Pharmaceutical preparations for oral use can be obtained by combiningthe active compounds with solid excipient, optionally grinding aresulting mixture, and processing the mixture of granules, after addingsuitable auxiliaries, if desired, to obtain tablets or dragee cores.Suitable excipients are, in particular, fillers such as sugars,including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations such as, for example, maize starch, wheat starch, ricestarch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxy-methylcellulose, and/orpolyvinyl pyrrolidone (PVP). If desired, disintegrating agents may beadded, such as the cross-linked polyvinyl pyrrolidone, agar, or alginicacid or a salt thereof such as sodium alginate.

Dragee cores are provided with suitable coating. For this purpose,concentrated sugar solutions may be used, which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers may be added.

The composition can include a buffer system, if desired. Buffer systemsare chosen to maintain or buffer the pH of compositions within a desiredrange. The term “buffer system” or “buffer” as used herein refers to asolute agent or agents which, when in a water solution, stabilize suchsolution against a major change in pH (or hydrogen ion concentration oractivity) when acids or bases are added thereto. Solute agent or agentswhich are thus responsible for a resistance or change in pH from astarting buffered pH value in the range indicated above are well known.While there are countless suitable buffers, potassium phosphatemonohydrate is a preferred buffer.

The final pH value of the pharmaceutical composition may vary within thephysiological compatible range. Necessarily, the final pH value is onenot irritating to human skin and preferably such that transdermaltransport of the active compound, i.e. CoQ10 is facilitated. Withoutviolating this constraint, the pH may be selected to improve CoQ10compound stability and to adjust consistency when required. In oneembodiment, the preferred pH value is about 3.0 to about 7.4, morepreferably about 3.0 to about 6.5, most preferably from about 3.5 toabout 6.0.

For preferred topical delivery vehicles the remaining component of thecomposition is water, which is necessarily purified, e.g., deionizedwater. Such delivery vehicle compositions contain water in the range ofmore than about 50 to about 95 percent, based on the total weight of thecomposition. The specific amount of water present is not critical,however, being adjustable to obtain the desired viscosity (usually about50 cps to about 10,000 cps) and/or concentration of the othercomponents. The topical delivery vehicle preferably has a viscosity ofat least about 30 centipoises.

Other known transdermal skin penetration enhancers can also be used tofacilitate delivery of CoQ10. Illustrative are sulfoxides such asdimethylsulfoxide (DMSO) and the like; cyclic amides such as1-dodecylazacycloheptane-2-one (Azone.™., a registered trademark ofNelson Research, Inc.) and the like; amides such as N,N-dimethylacetamide (DMA) N,N-diethyl toluamide, N,N-dimethyl formamide,N,N-dimethyl octamide, N,N-dimethyl decamide, and the like; pyrrolidonederivatives such as N-methyl-2-pyrrolidone, 2-pyrrolidone,2-pyrrolidone-5-carboxylic acid, N-(2-hydroxyethyl)-2-pyrrolidone orfatty acid esters thereof, 1-lauryl-4-methoxycarbonyl-2-pyrrolidone,N-tallowalkylpyrrolidones, and the like; polyols such as propyleneglycol, ethylene glycol, polyethylene glycol, dipropylene glycol,glycerol, hexanetriol, and the like; linear and branched fatty acidssuch as oleic, linoleic, lauric, valeric, heptanoic, caproic, myristic,isovaleric, neopentanoic, trimethyl hexanoic, isostearic, and the like;alcohols such as ethanol, propanol, butanol, octanol, oleyl, stearyl,linoleyl, and the like; anionic surfactants such as sodium laurate,sodium lauryl sulfate, and the like; cationic surfactants such asbenzalkonium chloride, dodecyltrimethylammonium chloride,cetyltrimethylammonium bromide, and the like; non-ionic surfactants suchas the propoxylated polyoxyethylene ethers, e.g., Poloxamer 231,Poloxamer 182, Poloxamer 184, and the like, the ethoxylated fatty acids,e.g., Tween 20, Myjr 45, and the like, the sorbitan derivatives, e.g.,Tween 40, Tween 60, Tween 80, Span 60, and the like, the ethoxylatedalcohols, e.g., polyoxyethylene (4) lauryl ether (Brij 30),polyoxyethylene (2) oleyl ether (Brij 93), and the like, lecithin andlecithin derivatives, and the like; the terpenes such as D-limonene,α.-pinene, β-carene, α-terpineol, carvol, carvone, menthone, limoneneoxide, α-pinene oxide, eucalyptus oil, and the like. Also suitable asskin penetration enhancers are organic acids and esters such assalicyclic acid, methyl salicylate, citric acid, succinic acid, and thelike.

In one embodiment, the present invention provides CoQ10 compositions andmethods of preparing the same. Preferably, the compositions comprise atleast about 1% to about 25% CoQ10 w/w. CoQ10 can be obtained from AsahiKasei N&P (Hokkaido, Japan) as UBIDECARENONE (USP). CoQ10 can also beobtained from Kaneka Q10 as Kaneka Q10 (USP UBIDECARENONE) in powderedform (Pasadena, Tex., USA). CoQ10 used in the methods exemplified hereinhave the following characteristics: residual solvents meet USP 467requirement; water content is less than 0.0%, less than 0.05% or lessthan 0.2%; residue on ignition is 0.0%, less than 0.05%, or less than0.2% less than; heavy metal content is less than 0.002%, or less than0.001%; purity of between 98-100% or 99.9%, or 99.5%. Methods ofpreparing the compositions are provided in the examples section below.

In certain embodiments of the invention, methods are provided fortreating or preventing an oncological disorder in a human by topicallyadministering Coenzyme Q10 to the human such that treatment orprevention occurs, wherein the human is administered a topical dose ofCoenzyme Q10 in a topical vehicle where Coenzyme Q10 is applied to thetarget tissue in the range of about 0.01 to about 0.5 milligrams ofcoenzyme Q10 per square centimeter of skin. In one embodiment, CoenzymeQ10 is applied to the target tissue in the range of about 0.09 to about0.15 mg CoQ10 per square centimeter of skin. In various embodiments,Coenzyme Q10 is applied to the target tissue in the range of about 0.001to about 5.0, about 0.005 to about 1.0, about 0.005 to about 0.5, about0.01 to about 0.5, about 0.025 to about 0.5, about 0.05 to about 0.4,about 0.05 to about 0.30, about 0.10 to about 0.25, or about 0.10 to0.20 mg CoQ10 per square centimeter of skin. In other embodiments,Coenzyme Q10 is applied to the target tissue at a dose of about 0.01,0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13,0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25,0.26, 0.27, 0.28, 0.29, 0.30, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37,0.38, 0.39, 0.40, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49or 0.5 mg CoQ10 per square centimeter of skin. In one embodiment,Coenzyme Q10 is applied to the target tissue at a dose of about 0.12 mgCoQ10 per square centimeter of skin It should be understood that rangeshaving any one of these values as the upper or lower limits are alsointended to be part of this invention, e.g., about 0.03 to about 0.12,about 0.05 to about 0.15, about 0.1 to about 0.20, or about 0.32 toabout 0.49 mg CoQ10 per square centimeter of skin.

In another embodiment of the invention, the Coenzyme Q10 is administeredin the form of a CoQ10 cream at a dosage of between 0.5 and 10milligrams of the CoQ10 cream per square centimeter of skin, wherein theCoQ10 cream comprises between 1 and 5% of Coenzyme Q10. In oneembodiment, the CoQ10 cream comprises about 3% of Coenzyme Q10. In otherembodiments, the CoQ cream comprises about 1%, 1.5%, 2%, 2.5%, 3%, 3.5%,4%, 4.5% or 5% of Coenzyme Q10. In various embodiments, the CoQ10 creamis administered at a dosage of about 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5,4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5 or 10milligrams of CoQ10 cream per square centimeter of skin. It should beunderstood that ranges having any one of these values as the upper orlower limits are also intended to be part of this invention, e.g.,between about 0.5 and about 5.0, about 1.5 and 2.5, or about 2.5 and 5.5mg CoQ10 cream per square centimeter of skin.

In another embodiment, the Coenzyme Q10 is administered in the form of aCoQ10 cream at a dosage of between 3 and 5 milligrams of the CoQ10 creamper square centimeter of skin, wherein the CoQ10 cream comprises between1 and 5% of Coenzyme Q10. In one embodiment, the CoQ10 cream comprisesabout 3% of Coenzyme Q10. In other embodiments, the CoQ10 creamcomprises about 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5% or 5% of CoenzymeQ10. In various embodiments, the CoQ10 cream is administered at a dosageof about 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1,4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9 or 5.0 milligrams of CoQ10 creamper square centimeter of skin. It should be understood that rangeshaving any one of these values as the upper or lower limits are alsointended to be part of this invention, e.g., between about 3.0 and about4.0, about 3.3 and 5.3, or about 4.5 and 4.9 mg CoQ10 cream per squarecentimeter of skin.

Certain aspects of the invention provide methods for treating orpreventing an oncological disorder in a human by topically administeringCoenzyme Q10 to the human such that treatment or prevention occurs,wherein the Coenzyme Q10 is topically applied one or more times per 24hours for six weeks or more.

Certain aspects of the invention provide methods for the preparation ofa Coenzyme Q10 cream 3% which includes the steps of preparing a Phase A,B, C, D and E and combining all the phases such that an oil-in-wateremulsion of 3% CoQ10 cream is formed.

In some embodiments, the Phase A ingredients include Alkyl C₁₂₋₁₅benzoate NF at 4.00% w/w, cetyl alcohol NF at 2.00% w/w, glycerylstearate/PEG-100 at 4.5% w/w and stearyl alcohol NF at 1.50% w/w whilethe Phase B ingredients include diethylene glycol monoethyl ether NF at5.00% w/w, glycerin USP at 2.00% w/w, propylene glycol USP at 1.50% w/w,phenoxyethanol NF at 0.475% w/w, purified water USP at 16.725% w/w andCarbomer Dispersion 2% at 40.00% w/w and the Phace C ingredients includelactic acid USP at 0.50% w/w, sodium lactate solution USP at 2.00% w/w,trolamine NF at 1.30% w/w, and purified water USP at 2.50% w/w.Furthermore in these embodiments the Phase D ingredients includetitanium dioxide USP at 1.00% w/w while the Phase E ingredients includeCoQ10 21% concentrate at 15% w/w.

In certain other embodiments, the Phase A ingredients includecapric/caprylic triglyceride at 4.00% w/w, cetyl alcohol NF at 2.00%w/w, glyceril stearate/PEG-100 at 4.5% and stearyl alcohol NF at 1.5%w/w while the Phase B ingredients include diethylene glycol monoethylether NF at 5.00% w/w, glycerin USP at 2.00% w/w, propylene glycol USPat 1.50% w/w, phenoxyethanol NF at 0.475% w/w, purified water USP at16.725% w/w and Carbomer Dispersion 2% at 40.00% w/w and the Phace Cingredients include lactic acid USP at 0.50% w/w, sodium lactatesolution USP at 2.00% w/w, trolamine NF at 1.30% w/w, and purified waterUSP at 2.50% w/w. Furthermore in these embodiments the Phase Dingredients include titanium dioxide USP at 1.00% w/w while the Phase Eingredients include CoQ10 21% concentrate at 15% w/w.

In certain embodiments of the invention, methods are provided for thepreparation of a Coenzyme Q10 cream 3% which include the steps of (1)adding the Phase A ingredients to a suitable container and heating to70-80 degrees C. in a water bath; (2) adding the Phase B ingredients,excluding the Carbomer Dispersion, to a suitable container and mixing toform a mixed Phase B; (3) placing the Phase E ingredients into asuitable container and melting them at 50-60 degrees C. using a waterbath to form a melted Phase E; (4) adding the Carbomer Dispersion to aMix Tank and heating to 70-80 degrees C. while mixing; (5) adding themixed Phase B to the Mix Tank while maintaining the temperature at 70-80degrees C.; (6) adding the Phase C ingredients to the Mix Tank whilemaintaining the temperature at 70-80 degrees C.; (7) adding the Phase Dingredients to the Mix Tank and then continue mixing and homogenizingthe contents of the Mix Tank; then (8) stopping the homogenization andcooling the contents of the Mix Tank to 50-60 degrees C.; then (9)discontinuing the mixing and adding the melted Phase E to the Mix Tankto form a dispersion; (10) mixing is then resumed until the dispersionis smooth and uniform; then (11) cooling the contents of the Mix Tank to45-50 degrees C.

In some other embodiments of the invention, a pharmaceutical compositioncomprising CoQ10 cream 3% is provided. The cream includes a phase Ahaving C₁₂₋₁₅ alkyl benzoate at 4.00% w/w of the composition, cetylalcohol at 2.00% w/w of the composition, stearyl alcohol at 1.5% w/w,glyceryl stearate and PEG-100 at 4.5% w/w; a phase B having glycerin at2.00% w/w, propylene glycol at 1.5% w/w, ethoxydiglycol at 5.0% w/w,phenoxyethanol at 0.475% w/w, a carbomer dispersion at 40.00% w/w,purified water at 16.725% w/w; a phase C having triethanolamine at1.300% w/w, lactic acid at 0.500% w/w, sodium lactate solution at 2.000%w/w, water at 2.5% w/w; a phase D having titanium dioxide at 1.000% w/w;and a phase E having CoQ10 21% concentrate at 15.000% w/w. In someembodiments the Carbomer Dispersion includes water, phenoxyethanol,propylene glycol and Carbomer 940.

In some other embodiments of the invention, a pharmaceutical compositioncomprising CoQ10 cream 3% is provided. The cream includes a phase Ahaving Capric/Caprylic triglyceride at 4.00% w/w of the composition,cetyl alcohol at 2.00% w/w of the composition, stearyl alcohol at 1.5%w/w, glyceryl stearate and PEG-100 at 4.5% w/w; a phase B havingglycerin at 2.00% w/w, propylene glycol at 1.5% w/w, ethoxydiglycol at5.0% w/w, phenoxyethanol at 0.475% w/w, a carbomer dispersion at 40.00%w/w, purified water at 16.725% w/w; a phase C having triethanolamine at1.300% w/w, lactic acid at 0.500% w/w, sodium lactate solution at 2.000%w/w, water at 2.5% w/w; a phase D having titanium dioxide at 1.000% w/w;and a phase E having CoQ10 21% concentrate at 15.000% w/w. In someembodiments the Carbomer Dispersion includes water, phenoxyethanol,propylene glycol and Carbomer 940.

In some other embodiments of the invention, a pharmaceutical compositioncomprising CoQ10 cream 1.5% is provided. The cream includes a phase Ahaving C₁₂₋₁₅ alkyl benzoate at 5.000% w/w, cetyl alcohol at 2.000% w/w,stearyl alcohol at 1.5% w/w, glyceryl stearate and PEG-100 stearate at4.500% w/w; a phase B having glycerin at 2.000% w/w, propylene at 1.750%w/w, ethoxydiglycol at 5.000% w/w, phenoxyethanol at 0.463% w/w, acarbomer dispersion at 50% w/w, and purified water at 11.377% w/w; aphase C having triethanolamine at 1.3% w/w, lactic acid at 0.400% w/w,sodium lactate solution at 2.000% w/w, and water at 4.210% w/w; a phaseD having titanium dioxide at 1.000% w/w; and a phase E having CoQ10 21%concentrate at 1.500% w/w.

In some other embodiments of the invention, a pharmaceutical compositioncomprising CoQ cream 1.5% is provided. The cream includes a phase Ahaving Capric/Caprylic triglyceride at 5.000% w/w, cetyl alcohol at2.000% w/w, stearyl alcohol at 1.5% w/w, glyceryl stearate and PEG-100stearate at 4.500% w/w; a phase B having glycerin at 2.000% w/w,propylene at 1.750% w/w, ethoxydiglycol at 5.000% w/w, phenoxyethanol at0.463% w/w, a carbomer dispersion at 50% w/w, and purified water at11.377% w/w; a phase C having triethanolamine at 1.3% w/w, lactic acidat 0.400% w/w, sodium lactate solution at 2.000% w/w, and water at4.210% w/w; a phase D having titanium dioxide at 1.000% w/w; and a phaseE having CoQ10 21% concentrate at 1.500% w/w. In some embodiments theCarbomer Dispersion includes water, phenoxyethanol and propylene glycol.

1. Combination Therapies

In certain embodiments, an environmental influencer of the inventionand/or pharmaceutical compositions thereof can be used in combinationtherapy with at least one other therapeutic agent, which may be adifferent environmental influencer and/or pharmaceutical compositionsthereof. The environmental influencer and/or pharmaceutical compositionthereof and the other therapeutic agent can act additively or, morepreferably, synergistically. In one embodiment, an environmentalinfluencer and/or a pharmaceutical composition thereof is administeredconcurrently with the administration of another therapeutic agent. Inanother embodiment, a compound and/or pharmaceutical composition thereofis administered prior or subsequent to administration of anothertherapeutic agent.

In one embodiment, the therapeutic methods of the invention compriseadditional agents. For example, in one embodiment, an additional agentfor use in the therapeutic methods of the invention of the invention isa chemotherapeutic agent.

Chemotherapeutic agents generally belong to various classes including,for example: 1. Topoisomerase II inhibitors (cytotoxic antibiotics),such as the antracyclines/anthracenediones, e.g., doxorubicin,epirubicin, idarubicin and nemorubicin, the anthraquinones, e.g.,mitoxantrone and losoxantrone, and the podophillotoxines, e.g.,etoposide and teniposide; 2. Agents that affect microtubule formation(mitotic inhibitors), such as plant alkaloids (e.g., a compoundbelonging to a family of alkaline, nitrogen-containing molecules derivedfrom plants that are biologically active and cytotoxic), e.g., taxanes,e.g., paclitaxel and docetaxel, and the vinka alkaloids, e.g.,vinblastine, vincristine, and vinorelbine, and derivatives ofpodophyllotoxin; 3. Alkylating agents, such as nitrogen mustards,ethyleneimine compounds, alkyl sulphonates and other compounds with analkylating action such as nitrosoureas, dacarbazine, cyclophosphamide,ifosfamide and melphalan; 4. Antimetabolites (nucleoside inhibitors),for example, folates, e.g., folic acid, fiuropyrimidines, purine orpyrimidine analogues such as 5-fluorouracil, capecitabine, gemcitabine,methotrexate and edatrexate; 5. Topoisomerase I inhibitors, such astopotecan, irinotecan, and 9-nitrocamptothecin, and camptothecinderivatives; and 6. Platinum compounds/complexes, such as cisplatin,oxaliplatin, and carboplatin. Exemplary chemotherapeutic agents for usein the methods of the invention include, but are not limited to,amifostine (ethyol), cisplatin, dacarbazine (DTIC), dactinomycin,mechlorethamine (nitrogen mustard), streptozocin, cyclophosphamide,carmustine (BCNU), lomustine (CCNU), doxorubicin (adriamycin),doxorubicin lipo (doxil), gemcitabine (gemzar), daunorubicin,daunorubicin lipo (daunoxome), procarbazine, mitomycin, cytarabine,etoposide, methotrexate, 5-fluorouracil (5-FU), vinblastine,vincristine, bleomycin, paclitaxel (taxol), docetaxel (taxotere),aldesleukin, asparaginase, busulfan, carboplatin, cladribine,camptothecin, CPT-II, lO-hydroxy-7-ethyl-camptothecin (SN38),dacarbazine, S—I capecitabine, ftorafur, 5′deoxyfluorouridine, UFT,eniluracil, deoxycytidine, 5-azacytosine, 5-azadeoxycytosine,allopurinol, 2-chloro adenosine, trimetrexate, aminopterin,methylene-10-deazaminopterin (MDAM), oxaplatin, picoplatin, tetraplatin,satraplatin, platinum-DACH, ormaplatin, CI-973, JM-216, and analogsthereof, epirubicin, etoposide phosphate, 9-aminocamptothecin,10,11-methylenedioxycamptothecin, karenitecin, 9-nitrocamptothecin, TAS103, vindesine, L-phenylalanine mustard, ifosphamidemefosphamide,perfosfamide, trophosphamide carmustine, semustine, epothilones A-E,tomudex, 6-mercaptopurine, 6-thioguanine, amsacrine, etoposidephosphate, karenitecin, acyclovir, valacyclovir, ganciclovir,amantadine, rimantadine, lamivudine, zidovudine, bevacizumab,trastuzumab, rituximab, 5-Fluorouracil, Capecitabine, Pentostatin,Trimetrexate, Cladribine, floxuridine, fludarabine, hydroxyurea,ifosfamide, idarubicin, mesna, irinotecan, mitoxantrone, topotecan,leuprolide, megestrol, melphalan, mercaptopurine, plicamycin, mitotane,pegaspargase, pentostatin, pipobroman, plicamycin, streptozocin,tamoxifen, teniposide, testolactone, thioguanine, thiotepa, uracilmustard, vinorelbine, chlorambucil, cisplatin, doxorubicin, paclitaxel(taxol) and bleomycin, and combinations thereof which are readilyapparent to one of skill in the art based on the appropriate standard ofcare for a particular tumor or cancer.

In another embodiment, an additional agent for use in the combinationtherapies of the invention is a biologic agent.

Biological agents (also called biologies) are the products of abiological system, e.g., an organism, cell, or recombinant system.Examples of such biologic agents include nucleic acid molecules (e.g.,antisense nucleic acid molecules), interferons, interleukins,colony-stimulating factors, antibodies, e.g., monoclonal antibodies,anti-angiogenesis agents, and cytokines. Exemplary biologic agents arediscussed in more detail below and generally belong to various classesincluding, for example: 1. Hormones, hormonal analogues, and hormonalcomplexes, e.g., estrogens and estrogen analogs, progesterone,progesterone analogs and progestins, androgens, adrenocorticosteroids,antiestrogens, antiandrogens, antitestosterones, adrenal steroidinhibitors, and anti-leuteinizing hormones; and 2. Enzymes, proteins,peptides, polyclonal and/or monoclonal antibodies, such as interleukins,interferons, colony stimulating factor, etc.

In one embodiment, the biologic is an interfereon. Interferons (IFN) area type biologic agent that naturally occurs in the body. Interferons arealso produced in the laboratory and given to cancer patients inbiological therapy. They have been shown to improve the way a cancerpatient's immune system acts against cancer cells.

Interferons may work directly on cancer cells to slow their growth, orthey may cause cancer cells to change into cells with more normalbehavior. Some interferons may also stimulate natural killer cells (NK)cells, T cells, and macrophages, which are types of white blood cells inthe bloodstream that help to fight cancer cells.

In one embodiment, the biologic is an interleukin. Interleukins (IL)stimulate the growth and activity of many immune cells. They areproteins (cytokines and chemokines) that occur naturally in the body,but can also be made in the laboratory.

Some interleukins stimulate the growth and activity of immune cells,such as lymphocytes, which work to destroy cancer cells.

In another embodiment, the biologic is a colony-stimulating factor.

Colony-stimulating factors (CSFs) are proteins given to patients toencourage stem cells within the bone marrow to produce more blood cells.The body constantly needs new white blood cells, red blood cells, andplatelets, especially when cancer is present. CSFs are given, along withchemotherapy, to help boost the immune system. When cancer patientsreceive chemotherapy, the bone marrow's ability to produce new bloodcells is suppressed, making patients more prone to developinginfections. Parts of the immune system cannot function without bloodcells, thus colony-stimulating factors encourage the bone marrow stemcells to produce white blood cells, platelets, and red blood cells.

With proper cell production, other cancer treatments can continueenabling patients to safely receive higher doses of chemotherapy.

In another embodiment, the biologic is an antibody. Antibodies, e.g.,monoclonal antibodies, are agents, produced in the laboratory, that bindto cancer cells.

When cancer-destroying agents are introduced into the body, they seekout the antibodies and kill the cancer cells. Monoclonal antibody agentsdo not destroy healthy cells. Monoclonal antibodies achieve theirtherapeutic effect through various mechanisms. They can have directeffects in producing apoptosis or programmed cell death. They can blockgrowth factor receptors, effectively arresting proliferation of tumorcells. In cells that express monoclonal antibodies, they can bring aboutanti idiotype antibody formation.

Examples of antibodies which may be used in the combination treatment ofthe invention include anti-CD20 antibodies, such as, but not limited to,cetuximab, Tositumomab, rituximab, and Ibritumomab. Anti-HER2 antibodiesmay also be used in combination with an environmental influencer for thetreatment of cancer. In one embodiment, the anti-HER2 antibody isTrastuzumab (Herceptin). Other examples of antibodies which may be usedin combination with an environmental influencer for the treatment ofcancer include anti-CD52 antibodies (e.g., Alemtuzumab), anti-CD-22antibodies (e.g., Epratuzumab), and anti-CD33 antibodies (e.g.,Gemtuzumab ozogamicin). Anti-VEGF antibodies may also be used incombination with an environmental influencer for the treatment ofcancer. In one embodiment, the anti-VEGF antibody is bevacizumab. Inother embodiments, the biologic agent is an antibody which is ananti-EGFR antibody e.g., cetuximab. Another example is theanti-glycoprotein 17-1A antibody edrecolomab.

In another embodiment, the biologic is a cytokine. Cytokine therapy usesproteins (cytokines) to help a subject's immune system recognize anddestroy those cells that are cancerous. Cytokines are produced naturallyin the body by the immune system, but can also be produced in thelaboratory. This therapy is used with advanced melanoma and withadjuvant therapy (therapy given after or in addition to the primarycancer treatment). Cytokine therapy reaches all parts of the body tokill cancer cells and prevent tumors from growing.

In another embodiment, the biologic is a fusion protein. For example,recombinant human Apo2L/TRAIL (Genentech) may be used in a combinationtherapy. Apo2/TRAIL is the first dual pro-apoptotic receptor agonistdesigned to activate both pro-apoptotic receptors DR4 and DR5, which areinvolved in the regulation of apoptosis (programmed cell death).

In one embodiment, the biologic is an antisense nucleic acid molecule.

As used herein, an “antisense” nucleic acid comprises a nucleotidesequence which is complementary to a “sense” nucleic acid encoding aprotein, e.g., complementary to the coding strand of a double-strandedcDNA molecule, complementary to an mRNA sequence or complementary to thecoding strand of a gene. Accordingly, an antisense nucleic acid canhydrogen bond to a sense nucleic acid.

In one embodiment, a biologic agent is an siRNA molecule, e.g., of amolecule that enhances angiogenesis, e.g., bFGF, VEGF and EGFR. In oneembodiment, a biologic agent that inhibits angiogenesis mediates RNAi.RNA interference (RNAi) is a post-transcriptional, targetedgene-silencing technique that uses double-stranded RNA (dsRNA) todegrade messenger RNA (mRNA) containing the same sequence as the dsRNA(Sharp, P. A. and Zamore, P. D. 287, 2431-2432 (2000); Zamore, P. D., etal. Cell 101, 25-33 (2000). Tuschl, T. et al. Genes Dev. 13, 3191-3197(1999); Cottrell T R, and Doering T L. 2003. Trends Microbiol. 11:37-43;Bushman F.2003. MoI Therapy. 7:9-10; McManus M T and Sharp P A. 2002.Nat Rev Genet. 3.737-47). The process occurs when an endogenousribonuclease cleaves the longer dsRNA into shorter, e.g., 21- or22-nucleotide-long RNAs, termed small interfering RNAs or siRNAs. Thesmaller RNA segments then mediate the degradation of the target mRNA.Kits for synthesis of RNAi are commercially available from, e.g. NewEngland Biolabs or Ambion. In one embodiment one or more chemistries foruse in antisense RNA can be employed in molecules that mediate RNAi.

The use of antisense nucleic acids to downregulate the expression of aparticular protein in a cell is well known in the art (see e.g.,Weintraub, H. et al., Antisense RNA as a molecular tool for geneticanalysis, Reviews—Trends in Genetics, Vol. 1(1) 1986; Askari, F. K. andMcDonnell, W. M. (1996) N. Eng. J. Med. 334:316-318; Bennett, M. R. andSchwartz, S. M. (1995) Circulation 92:1981-1993; Mercola, D. and Cohen,J. S. (1995) Cancer Gene Ther. 2:47-59; Rossi, J J. (1995) Br. Med.Bull. 51.217-225; Wagner, R. W. (1994) Nature 372:333-335). An antisensenucleic acid molecule comprises a nucleotide sequence that iscomplementary to the coding strand of another nucleic acid molecule(e.g., an mRNA sequence) and accordingly is capable of hydrogen bondingto the coding strand of the other nucleic acid molecule. Antisensesequences complementary to a sequence of an mRNA can be complementary toa sequence found in the coding region of the mRNA, the 5′ or 3′untranslated region of the mRNA or a region bridging the coding regionand an untranslated region (e.g., at the junction of the 5′ untranslatedregion and the coding region). Furthermore, an antisense nucleic acidcan be complementary in sequence to a regulatory region of the geneencoding the mRNA, for instance a transcription initiation sequence orregulatory element. Preferably, an antisense nucleic acid is designed soas to be complementary to a region preceding or spanning the initiationcodon on the coding strand or in the 3′ untranslated region of an mRNA.

Given the coding strand sequences of a molecule that enhancesangiogenesis, antisense nucleic acids of the invention can be designedaccording to the rules of Watson and Crick base pairing. The antisensenucleic acid molecule can be complementary to the entire coding regionof the mRNA, but more preferably is an oligonucleotide which isantisense to only a portion of the coding or noncoding region of themRNA. For example, the antisense oligonucleotide can be complementary tothe region surrounding the translation start site of the mRNA. Anantisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25,30, 35, 40, 45 or 50 nucleotides in length.

An antisense nucleic acid of the invention can be constructed usingchemical synthesis and enzymatic ligation reactions using proceduresknown in the art. For example, an antisense nucleic acid (e.g., anantisense oligonucleotide) can be chemically synthesized using naturallyoccurring nucleotides or variously modified nucleotides designed toincrease the biological stability of the molecules or to increase thephysical stability of the duplex formed between the antisense and sensenucleic acids, e.g., phosphorothioate derivatives and acridinesubstituted nucleotides can be used. Examples of modified nucleotideswhich can be used to generate the antisense nucleic acid include5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyl uracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N-6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine. To inhibit expression in cells, one or moreantisense oligonucleotides can be used. Alternatively, the antisensenucleic acid can be produced biologically using an expression vectorinto which a nucleic acid has been subcloned in an antisense orientation(i.e., RNA transcribed from the inserted nucleic acid will be of anantisense orientation to a target nucleic acid of interest, describedfurther in the following subsection).

In yet another embodiment, the antisense nucleic acid molecule of theinvention is an a-anomeric nucleic acid molecule. An a-anomeric nucleicacid molecule forms specific double-stranded hybrids with complementaryRNA in which, contrary to the usual a-units, the strands run parallel toeach other (Gaultier et al. (1987) Nucleic Acids. Res. 15:6625-6641).The antisense nucleic acid molecule can also comprise a2′-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res.15:613-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987) FEBSLett. 215:327-330).

In another embodiment, an antisense nucleic acid of the invention is acompound that mediates RNAi. RNA interfering agents include, but are notlimited to, nucleic acid molecules including RNA molecules which arehomologous to the target gene or genomic sequence, “short interferingRNA” (siRNA), “short hairpin” or “small hairpin RNA” (shRNA), and smallmolecules which interfere with or inhibit expression of a target gene byRNA interference (RNAi). RNA interference is a post-transcriptional,targeted gene-silencing technique that uses double-stranded RNA (dsRNA)to degrade messenger RNA (mRNA) containing the same sequence as thedsRNA (Sharp, P. A. and Zamore, P. D. 287, 2431-2432 (2000); Zamore, P.D., et al. Cell 101, 25-33 (2000). Tuschl, T. et al. Genes Dev. 13,3191-3197 (1999)). The process occurs when an endogenous ribonucleasecleaves the longer dsRNA into shorter, 21- or 22-nucleotide-long RNAs,termed small interfering RNAs or siRNAs. The smaller RNA segments thenmediate the degradation of the target mRNA. Kits for synthesis of RNAiare commercially available from, e.g. New England Biolabs and Ambion. Inone embodiment one or more of the chemistries described above for use inantisense RNA can be employed.

Nucleic acid molecules encoding molecules that, e.g., inhibitangiogenesis, may be introduced into the subject in a form suitable forexpression of the encoded protein in the cells of the subject may alsobe used in the methods of the invention. Exemplary molecules thatinhibit angiogenesis include, but are not limited to, TSP-I, TSP-2,IFN-g, IFN-a, angiostatin, endostatin, tumastatin, canstatin, VEGI,PEDF, vasohibin, and the 16 kDa fragment of prolactin 2-Methoxyestradiol(see, Kerbel (2004) J. Clin Invest 114:884, for review).

For example, a full length or partial cDNA sequence is cloned into arecombinant expression vector and the vector is transfected into a cellusing standard molecular biology techniques. The cDNA can be obtained,for example, by amplification using the polymerase chain reaction (PCR)or by screening an appropriate cDNA library. The nucleotide sequences ofthe cDNA can be used for the design of PCR primers that allow foramplification of a cDNA by standard PCR methods or for the design of ahybridization probe that can be used to screen a cDNA library usingstandard hybridization methods. Following isolation or amplification ofthe cDNA, the DNA fragment is introduced into a suitable expressionvector.

Exemplary biologic agents for use in the methods of the inventioninclude, but are not limited to, gefitinib (Iressa), anastrazole,diethylstilbesterol, estradiol, premarin, raloxifene, progesterone,norethynodrel, esthisterone, dimesthisterone, megestrol acetate,medroxyprogesterone acetate, hydroxyprogesterone caproate,norethisterone, methyltestosterone, testosterone, dexamthasone,prednisone, Cortisol, solumedrol, tamoxifen, fulvestrant, toremifene,aminoglutethimide, testolactone, droloxifene, anastrozole, bicalutamide,flutamide, nilutamide, goserelin, flutamide, leuprolide, triptorelin,aminoglutethimide, mitotane, goserelin, cetuximab, erlotinib, imatinib,Tositumomab, Alemtuzumab, Trastuzumab, Gemtuzumab, Rituximab,Ibritumomab tiuxetan, Bevacizumab, Denileukin diftitox, Daclizumab,interferon alpha, interferon beta, anti-4-IBB, anti-4-IBBL, anti-CD40,anti-CD 154, anti-OX40, anti-OX40L, anti-CD28, anti-CD80, anti-CD86,anti-CD70, anti-CD27, anti-HVEM, anti-LIGHT, anti-GITR, anti-GITRL,anti-CTLA-4, soluble OX40L, soluble 4-IBBL, soluble CD154, solubleGITRL, soluble LIGHT, soluble CD70, soluble CD80, soluble CD86, solubleCTLA4-Ig, GVAX®, and combinations thereof which are readily apparent toone of skill in the art based on the appropriate standard of care for aparticular tumor or cancer. The soluble forms of agents may be made as,for example fusion proteins, by operatively linking the agent with, forexample, Ig-Fc region.

It should be noted that more than one additional agent, e.g., 1, 2, 3,4, 5, may be administered in combination with an environmentalinfluencer. For example, in one embodiment two chemotherapeutic agentsmay be administered in combination with an environmental influencer. Inanother embodiment, a chemotherapeutic agent, a biologic agent, and anenvironmental influencer may be administered.

Various forms of the biologic agents may be used. These include, withoutlimitation, such forms as proform molecules, uncharged molecules,molecular complexes, salts, ethers, esters, amides, and the like, whichare biologically activated when implanted, injected or otherwiseinserted into the tumor.

This invention is further illustrated by the following examples whichshould not be construed as limiting. The contents of all references andpublished patents and patent applications cited throughout theapplication are hereby incorporated by reference.

EXEMPLIFICATION OF THE INVENTION

The invention now being generally described, it will be more readilyunderstood by reference to the following examples, which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention,as one skilled in the art would recognize from the teachings hereinabove and the following examples, that other assays, cell types, agents,constructs, or data analysis methods, all without limitation, can beemployed, without departing from the scope of the invention as claimed.

The contents of any patents, patent applications, patent publications,or scientific articles referenced anywhere in this application areherein incorporated in their entirety.

The practice of the present invention will employ, where appropriate andunless otherwise indicated, conventional techniques of cell biology,cell culture, molecular biology, transgenic biology, microbiology,virology, recombinant DNA, and immunology, which are within the skill ofthe art. Such techniques are described in the literature. See, forexample, Molecular Cloning: A Laboratory Manual, 3rd Ed., ed. bySambrook and Russell (Cold Spring Harbor Laboratory. Press: 2001); thetreatise, Methods In Enzymology (Academic Press, Inc., N.Y.); UsingAntibodies, Second Edition by Harlow and Lane, Cold Spring Harbor Press,New York, 1999; Current Protocols in Cell Biology, ed. by Bonifacino,Dasso, Lippincott-Schwartz, Harford, and Yamada, John Wiley and Sons,Inc., New York, 1999; and PCR Protocols, ed. by Bartlett et al., HumanaPress, 2003.

Example 1 Identification of CoQ10 as a MIM

In order to evaluate CoQ10 as a potential MIM, CoQ10 in oxidized formwas exogenously added to a panel of cell lines, including both cancercell lines and normal control cell lines, and the changes induced to thecellular microenvironment profile for each cell line in the panel wereassessed. Changes to cell morphology/physiology, and to cellcomposition, including both mRNA and protein levels, were evaluated andcompared for the diseased cells as compared to normal cells. The resultsof these experiments identified CoQ10 and, in particular, the oxidizedform of CoQ10, as a MIM.

In a first set of experiments, changes to cell morphology/physiologywere evaluated by examining the sensitivity and apoptotic response ofcells to CoQ10. A panel of skin cell lines including a control celllines (primary culture of keratinocytes and melanocytes) and severalskin cancers cell lines (SK-MEL-28, a non-metastatic skin melanoma;SK-MEL-2, a metastatic skin melanoma; or SCC, a squamous cell carcinoma;PaCa2, a pancreatic cancer cell line; or HEP-G2, a liver cancer cellline) were treated with various levels of Coenzyme Q10. The results ofthese experiments demonstrated that the cancer cell lines exhibited analtered dose dependent response as compared to the control cell lines,with an induction of apoptosis and cell death in the cancer cells only.Exemplary experiments are described in detail in Example 3 below.

Assays were next employed to assess changes in the composition of thecell following treatment with CoQ10. Changes in gene expression at themRNA level were analyzed using Real-Time PCR array methodology.Exemplary experiments are described in detail in Examples 6 and 9-13below. In complementary experiments, changes in gene expression at theprotein level were analyzed by using antibody microarray methodology,2-dimensional gel electrophoresis followed by protein identificationusing mass spectrometry characterization, and by western blot analysis.Exemplary experiments are described in detail below in Examples 4, 7 and8, respectively. The results from these assays demonstrated thatsignificant changes in gene expression, both at the mRNA and proteinlevels, were induced in the cell lines examined due to the addition ofthe oxidized form of CoQ10. Genes modulated by CoQ10 treatment werefound to be clustered into several cellular pathways, includingapoptosis, cancer biology and cell growth, glycolysis and metabolism,molecular transport, and cellular signaling.

Experiments were carried out to confirm the entry of CoQ10 into cellsand to determine the level and form of CoQ10 present in the cells. Inparticular, the level of Coenzyme Q10, as well as the form of CoQ10(i.e., oxidized or reduced), present in the mitochondria was determinedby analyzing mitochondrial enriched preparations from cells treated withCoQ10. The level of Coenzyme Q10 present in the mitochondria wasconfirmed to increase in a time and dose dependent manner with theaddition of exogenous Q10. In a surprising and unexpected result, CoQ10was determined to be present in the mitochondria primarily in oxidizedform. In addition, changes in levels of proteins from mitochondriaenriched samples were analyzed by using 2-D gel electrophoresis andprotein identification by mass spectrometry characterization. Theresults from these experiments demonstrated that the levels of theoxidized form of CoQ10 in the mitochondria over the time course examinedcorrelated with a wide variety of cellular changes, as evidenced by themodulation of mRNA and protein levels for specific proteins related tometabolic and apoptotic pathways. Exemplary experiments are described indetail in Example 5 below.

The results described by Applicants herein identified the endogenousmolecule CoQ10 and, in particular, the oxidized form of CoQ10, as a MIM.For example, the results identified CoQ10 as a MIM, since CoQ10 wasobserved to induce changes in gene expression at both the mRNA andprotein level. The results identified CoQ10 as having multidimensionalcharacter, since CoQ10 induced differential changes in cellmorphology/physiology and cell composition (e.g., differential changesin gene expression at both the mRNA and protein level), in a diseasestate (e.g., cancer) as compared to a normal (e.g., non-cancerous)state. Moreover, the results identified CoQ10 as having multidimensionalcharacter in that CoQ10 was capable of entering a cell, and thusexhibited both therapeutic and carrier effects.

Example 2 Methods for Identifying Disease Relevant Processes andBiomarkers for Oncological Disorders

From the cell based assays in which cell lines were treated with amolecule of interest, the differences in treated vs non-treated cells isevaluated by mRNA arrays, protein antibody arrays, and 2D gelelectrophoresis. The proteins identified from comparative sampleanalysis to be modulated by the MIM or Epi-shifter, are evaluated from aSystems Biology perspective with pathway analysis (Ingenuity IPAsoftware) and a review Of the known literature. Proteins identified aspotential therapeutic or biomarker targets are submitted to confirmatoryassays such as Western blot analysis, siRNA knock-down, or recombinantprotein production and characterization methods.

Materials and Methods for Examples 3-8 Coenzyme Q10 Stock

A 500 μM Coenzyme Q10 (5% isopropanol in cell growth media) was preparedas follows. A 10 mL 500 μM Coenzyme Q10 stock was made fresh every time.

Molecular Weight: 863.34

(0.0005 mol/L)(0.010 L)(863.34 g/mol)=0.004317 g

To make 10 mL of 500 μM stock, 4.32 mg Coenzyme Q10 was weighted out ina 15 mL falcon tube, and 500 μL-isopropanol was added. The solution waswarmed in a 50-60° C. water bath while swirling to dissolve completely.To this solution, 9.5 mL of media (the same media in which the cells aregrown) was added.

Cell Culture

Cells were obtained from the American Type Culture Collection or Gibco.Cells were grown in DMEM/F-12 media supplemented with 5% fetal bovineserum, 0.25 ug/mL Amphotericin, 100 ug/mL Streptomycin, and 100 U mL-1penicillin. Cells were maintained in an atmosphere of 95% air and 5% CO2at 37 degrees C.

Coenzyme Q10 Treatment and Total Protein Isolation

Cells were grown to 85% confluency prior to exposure with Q10.Supplemented media was conditioned with Q10 to 50 and 100 micro molarconcentrations. Flasks were treated with control, 50 μM Q10, and 100 μMQ10 in triplicate. Protein was isolated from the treated and controlflask after 4, 8, 12, and 24 hours. For isolation of proteins, cellswere washed three times with 5 mL of ice cold PBS at a pH of 7.4. Thecells were then scraped in 3 mL of PBS, pelleted by centrifuge, andre-suspended in a lysis buffer at pH 7.4 (80 mM TRIS-HCl, 1% SDS, withprotease and phosphotase inhibitors). Protein concentrations werequantified using the BCA method.

Cell Lines

The cell lines listed below were propagated and a cell bank establishedfor each. Large scale production of cells for various assays wereperformed and the material harvested for analysis. In general, when acell specific media was not required for maintenance of cell lines, themedia used for cell growth was DMEMF-12 with 5% serum. Cells weretypically grown to 75-80% confluence (clear spacing) prior to splittingand use in cell assays and standard practice methods followed. Thefollowing cell lines were established for experiments:

-   -   SK-MEL-28 (non-metastatic skin melanoma)    -   SK-MEL-2 (metastatic skin melanoma)    -   HEKa (kerantinocytes, skin control)    -   HEMa (melanocyte, skin control)    -   nFIB (neonatal fibroblasts)    -   HEP-G2 (liver cancer) [SBH cell line]    -   SkBr-3 (breast cancer, Her2 overexpressed)    -   MCF-7 (breast cancer, p53 mutation)    -   PC-3 (prostate cancer) [SBH cell line]    -   SkBr-3 (human breast adenocarcinoma)    -   NCI-ES-0808    -   SCC (squamous cell carcinoma)    -   PaCa-2    -   NIH-3T3

Cell Culture:

Cells were obtained for the American Type Culture Collection or Gibco.Cells were grown in DMEM/F-12 media supplemented with 5% fetal bovineserum, 0.25 ug/mL Amphotericin, 100 ug/mL Streptomycin, and 100 U mL-1penicillin. Cells were maintained in an atmosphere of 95% air and 5% CO2at 37 degrees C.

Skin malignant melanoma SK-MEL28 cells were grown and maintained inDMEM/F12 with Glutamax (Invitrogen, Carlsbad Calif.) supplemented with5% FBS, amphotericin and penicillin/streptomycin. Cells were grown at37° C. with 5% CO2. Details of additional cell line and growthconditions are outlined in the table below.

TABLE 1 Cell lines analyzed for sensitivity to Q10. Cell LineDescription Growth Conditions PaCa2 Pancreatic DMEM/F12 with Glutamax +10% Carcinoma FBS, 2.5% Horse Serum, amphotericin,penicillin/streptomycin. HepG2 Hepatocellular MEM with Earles Saltssupplemented Carcinoma with 10% FBS, amphotericin,penicillin/streptomycin, sodium pyruvate and non-essential amino acids.PC3 Prostate DMEM/F12 with Glutamax, supplemented Adenocarcinoma with 5%FBS, amphotericin and penicillin/streptomycin. SKBr3 Breast CancerDMEM/F12 with Glutamax supplemented with 5% FBS and amphotericin,penicillin/streptomycin. MCF-7 Breast Cancer DMEM/F12 with Glutamaxsupplemented with 5% FBS and amphotericin, penicillin/streptomycin.

Q10 Treatment of SKMEL28 Cells:

SK-MEL28 cells were treated with 100 μM Q10 or the control vehicle. Theformulation of the Q10 was as follows. In a 15 mL capped tube, 4.32 mgof Q10 (supplied by Cytotech) was transferred and then dissolved by theaddition of 500 μL of isopropanol. The resulting solution was warmed ina 65° C. water bath and vortexed at high speed. The Q10/isopropanolsolution was made to a volume of 10 mL with the addition of equilibratedcell culture media. The stock solution was then vortexed to ensuremaximum solubility of Q10. The stock solution was diluted (2 mL of stockwith 8 mL of media) to obtain a final concentration of 100 μM Q10. Forthe control vehicle, 9.5 mL of media was added to 500 μL of isopropanol.The control stock was further diluted (2 mL of stock) with 8 mL ofmedia. Cells were harvested 6, 16, 24, 48 or 72 hours after the start ofthe treatment.

Q10 Treatment of SCC Cells:

SCC cells were treated with 100 μM Q10 (prepared as described above)either for 6 hours or 24 hours. The control cells were untreated cells.Cells were harvested and pelleted at the different times after treatmentand the pellets were flash frozen and stored at −80° C. until the RNAwas isolated at XTAL as described below.

RNA Isolation:

Cells were lysed for RNA isolation at different treatment times usingthe RNeasy Mini kit (Qiagen, Inc., Valencia Calif.) kit following themanufacturer's instructions. RNA was quantified by measuring OpticalDensity at 260 nm.

First Strand Synthesis:

First strand cDNA was synthesized from 1 μg of total RNA using the RT2First Strand Synthesis kit (SABiosciences, Frederick Md.) as permanufacturer's recommendations.

Real-Time PCR:

Products from the first strand synthesis were diluted with water, mixedwith the SYBR green master mix (SABiosciences, Frederick Md.) and loadedonto PCR arrays. Real time PCR was run on the PCR Arrays (ApoptosisArrays, Diabetes Arrays, Oxidative stress and Antioxidant defense Arraysand Heat Shock Protein Arrays.) (SABiosciences, Frederick Md.) on aBiorad CFX96.

Determining Cell Line Sensitivity to Coenzyme Q10 by Nexin Assay forApoptosis:

The percentage of cells in early and late apoptosis was quantifiedfollowing 24 hours of Coenzyme Q10 treatment. Early and late apoptosiswas used as a marker to understand the differences in sensitivity ofvarious cancer cell lines to Coenzyme Q10. The different cell linestested were PaCa2, HepG2, PC-3, SKBr3, MCF-7 and SK-MEL28. Cells wereallowed to adhere overnight in 96-well plates. These cells were treatedwith either control vehicle, 50 μM Q10 or 100 μM Coenzyme Q10. After 24hours, the presence of apoptotic cells was estimated on a PCA96 flowcytometer (Guava Technologies, Hayward, Calif.). In addition, some cellswere treated with 4 μM Staurosporine for 2 hours as a positive controlfor apoptosis. Cells were first washed with PBS and detached with 50 μLof Accumax (Innovative Cell Technologies, San Diego, Calif.) at roomtemperature. The dissociation was stopped by addition of culture mediumcontaining 1% Pluronic F-68 (Sigma-Aldrich, St. Louis, Mo.). Then 100 μLof Nexin reagent (Guava Technologies, Hayward, Calif.) was added to eachof the wells. After 20 minutes of incubation in the dark, the assay wasperformed in low binding plates to minimize reattachment of cells to thesubstrate. The Nexin Reagent contains two dyes: Annexin-V-PE whichdetects phosphotidyl serine on the outside of a cell; a characteristicof early apoptotic cells. The second dye, 7-AAD permeates only lateapoptotic cells while being excluded from live (healthy) and earlyapoptotic cells. The percentage of four populations of cells; live,early apoptotic, late apoptotic and debris was determined using theCytosoft 2.5.7 software (Guava Technologies, Hayward, Calif.).

Immunoblotting

Approximately 50 μg of protein were assayed per sample byimmunoblotting. All treatments were run in triplicate with controls.Proteins were separated on 12% TRIS-HCl gels, transferred viaelectrophoresis to nitro-cellulose membranes and blocked using a 5% milkand TBST solution prior to incubation with primary antibodies. Theprimary antibodies were incubated overnight at 4 degrees C. in a 5% BSAand TBST solution. Secondary antibodies were incubated for one hour at 4degrees. All antibodies were purchased from Cell Signaling Technology.Antibodies were used at a ratio of 1:1000, with the exception of βActinat a ratio of 1:5000. Blots were developed and results were quantifiedusing the NIH Java based densitometer analysis software Image J. Allblots were also probed for and normalized to their respective Actinexpression.

Two-Dimensional Electrophoresis

Before isoelectric focusing (IEF), samples were solubilized in 40 mMTris, 7 M urea, 2 M thiourea, and 1% C7 zwitterionic detergent, reducedwith tributylphosphine, and alkylated with 10 mM acrylamide for 90 minat room temperature. After the sample was run through a 10-kDa cutoffAmicon Ultra device with at least 3 volumes of the resuspension buffer,consisting of 7 M urea, 2 M thiourea, and 2% CHAPS to reduce theconductivity of the sample. One hundred micrograms of protein weresubjected to IEF on 11-cm pH 3 to 10, pH 4 to 7 or pH 6 to 11immobilized pH gradient strips (GE, Amersham, USA) to 100,000 voltshour. After IEF, immobilized pH gradient strips were equilibrated in 6 Murea, 2% SDS, 50 mM Tris-acetate buffer, pH 7.0, and 0.01% bromphenolblue and subjected to SDS-polyacrylamide gel electrophoresis on 8 to 16%Tris-HCl Precast Gel, 1 mm (Bio-Rad, USA). The gels were run induplicate. They were either fixed, stained in SYPRO Ruby, 80 mL/gel(Invitrogen, USA) and imaged on Fuji FLA-5100 laser scanner ortransferred onto PVDF membrane.

Additional information was obtained for a control sample to test theutility of protein identification through the use of methods thatutilize dPC (Protein Forest Inc.) selective pI fractionation, followedby trypsin digestion of the dPC plug with mass spec identification andsemi-quantization (Nanomate or LC/LTQ/MS). The dPC analysis performedwith a control sample demonstrated its utility in identifying a largesubset of proteins. The materials produced during the studies werearchived so that they may be utilized as a resource should the futureneed arise

2D Gel Image Analysis:

Analysis of all gel images was performed using Progenesis Discovery andPro (Nonlinear Dynamics Inc., Newcastle upon Tyne, UK). After spotdetection, matching, background subtraction, normalization, andfiltering, data for SYPRO Ruby gel images was exported. Pairwisecomparisons between groups were performed using the Student's t test inProgenesis Discovery to identify spots whose expression wassignificantly altered (p>0.05).

Antibody Array:

An antibody microarray (Panorama XP725 Antibody Array, Sigma) wasutilized to screen over 700 protein antibodies to assess changes at theprotein concentration level in Q10 treated cells (SK-MEL-28, SCC). Theexpression of a protein in a cell extract is detected when it is boundby a corresponding antibody spotted on the slide. Prior to binding, theproteins are directly labeled with a fluorescent dye which is used forfluorescent visualization and quantitative analysis. The array is usedfor comparing protein expression profiles of two samples (test versusreference samples), each labeled with a different CyDye (Cy3 or Cy5) andthe two samples are applied simultaneously at equal proteinconcentrations on the array. Fluorescent signal intensity for eachsample is then recorded individually at the wavelength corresponding tothe dye label of the sample and compared.

High doses of Coenzyme Q10 regulates expression of genes involved in theapoptotic, diabetic and oxidative stress pathways in cultured SKMEL-28cells.

Experimental details: SKMEL-28 cells (ATCC Catalog #HTB-72) are nonmetastatic, skin melanoma cells that were cultured in DMEM-F12containing Glutamax (Invitrogen Cat#10565-042) supplemented with 5% FBS,Penicillin, Streptomycin and Amphotericin, were treated with the vehicleor 100 uM Coenzyme Q10 for varying amounts of time. Any changes in geneexpression consequent to Coenzyme Q10 treatment were quantified usingReal time PCR Arrays (Apoptosis Cat #PAHS-12, Diabetes Cat #PAHS-023 andOxidative Stress Cat #PAHS-065). (SABiosciences, Frederick, Md.).

A stock concentration of 500 uM Coenzyme Q10 was prepared by dissolving4.32 mg in 500 ul of isopropanol which was further diluted to 10 ml byaddition of media. Alternate vortexing and heating to 65° C. dissolvedthe Coenzyme Q10. 2 ml of the stock solution was diluted to 10 ml withmedia to get a 100 uM Q10 containing media that was used to treat cells.A vehicle was prepared in parallel with a similar protocol except thatthe Coenzyme Q10 was not added.

SKMEL-28 cells were plated at a density of 1×10⁵ cells/well in a 6-wellplate. After 24 hours, when cells had attached and were at 50%confluence, either the vehicle or 100 uM Q10 was added. Cells wereharvested by at 6, 16, 24, 48 or 72 hours after Q10 treatment while thevehicle treated cells were harvested after 24 hours. Cells were lysedfor RNA isolation at different treatment times using the RNeasy Mini kit(Qiagen, Inc., Valencia Calif. Cat #74104) kit following themanufacturer's instructions using a spin column and on-column DNasetreatment. RNA was quantified by measuring absorbance at 260 nm.

Real time PCR was preceded by first strand cDNA synthesis using 0.4-lugof total RNA as the template using the RT2 First Strand Synthesis kit(SABiosciences, Frederick Md. Cat#C-03) with a genomic DNA eliminationstep as per manufacturer's recommendations. Products from the firststrand synthesis were diluted with water, mixed with the SYBR greenmaster mix (SABiosciences, Frederick Md. Cat#PA-010-12) and loaded ontoPCR arrays that contain primer assays for 84 different genes linkedwithin a common pathway, 5 housekeeping genes used for normalization,reverse transcription and PCR controls. Real time PCR was run on aBiorad Cfx96. The amplification was initiated with a hot start toactivate the enzyme, followed by 40 cycles each of (95° C.-15 seconddenaturation step and 60° C.-1 minute annealing and extension step)followed by a melting curve program. Ct values, the output from the PCRthermocycler for all treatment groups were organized on an excelspreadsheet and loaded onto the comparative analysis software availableat www dot sabiosciences dot corn slash per slash arrayanalysis dot php.

Purification of Mitochondria Enriched Samples:

Experimental details: SKMEL-28, NCI-ES0808 and NIH-3T3 cells that weretreated with 100 μM Q10 for 24 or 48 hours along with cells that wereharvested at t=0 were harvested by washing and scraping from T160flasks. Cells were centrifuged, pelleted, flash frozen and stored at−80° C. until the mitochondria were isolated. Cell pellets were thawed,resuspended and ruptured in Dounce homogenizer. The homogenate wascentrifuged and mitochondria were isolated using reagents and theprotocol recommended by the Mitochondria Isolation kit for Culturedcells (MitoSciences, Eugene Oreg., Cat #MS852). The mitochondrialfraction was aliquoted and stored at −80° C.

Coenzyme Q10 and Ubiquinol-10 Quantification Method:

A method for the simultaneous determination of Coenzyme Q10 (Q10) andthe reduced form ubiquinol-10 (Q10H2) was implemented based upon arecently published method (Ruiz-Jimenez, 2007, J. Chromatogr. A, 1175,242-248) through the use of LC-MS/MS with electrospray ionization (ESI)in the positive ion mode. The highly selective identification andsensitive quantitation of both Q10 and Q10H2 is possible, along with theidentification of other selected lipids. An aliquot of the mitochondrialenriched samples from SK-MEL-28 treated with 100 μM Q10 was subjected toa conventional pre-treatment based on protein precipitation (100 μl ofpacked cells sonicated in 300 μl of 1-propanol), liquid-liquidextraction (add 100 μl of water to supernatant and extract X3 with 200μl of n-hexane), evaporation of combined hexane extracts to dryness andreconstitution in 50 μl of 95:5 methanol/hexane (v/v). Analysis was byLC-MS/MS on a Waters Quattro II triple quadrupole mass spectrometer witha Prism RP 1×100 mm, 5 μm particle size column (Keystone Scientific).Isocratic elution with 4 mM ammonium formate in 20% isopropyl alcohol80% methanol at a flow rate of 50 μl/min. Ten μl of each sample wasinjected. MRM analysis was performed using m/z 882.7>197.00 (Q10H2) andm/z 880.80>197.00 (Q10) transitions with cone voltage of 40 andcollision energy of 30.

Example 3 Sensitivity of Cell Lines to CoQ10

A number of cell lines were tested for their sensitivity to Q10 after 24hours of application by using a reagent (Nexin reagent) that contains acombination of two dyes, 7AAD and Annexin-V-PE. The 7AAD dye will enterinto cells with permeabilized cell membranes; primarily those cells thatare in late apoptosis. Annexin-V-PE is a dye that binds to Phosphotidylserine, which is exposed on the outer surface of the plasma membrane inearly apoptotic cells. The Nexin reagent thus can be used todifferentiate between different populations of apoptotic cells in a flowcytometer.

PaCa2 cells showed an increase in both early and late apoptotic cells(between 5-10% of gated cells) with 50 μM Q10 and 100 μM Q10 after 24hours of Q10 application. PC-3 cells also showed an increase in bothearly and late apoptotic population with 50 μM and 100 μM Q10, althoughthe increase was less when compared to PaCa2 cells. MCF-7 and SK-MEL28cells showed an increase only in early apoptotic population with 50 μMand 100 μM Q10. HepG2 cells were also sensitive to 50 μM Q10 treatment,where there was an increase of about 20% of the gated populated in thelate apoptotic and early apoptotic stages. SKBr3 was the only cell linetested that did not show any significant increases of early and lateapoptosis with either 50 μM or 100 μM Q10 treatment. The results aredepicted in FIGS. 1-6.

To provide additional confirmation that Q10 treatment causes anapoptotic response in HepG2 liver cancer cells, a second apoptosis assaywas evaluated using the ApoStrand™ ELISA based method that measuressingle-stranded DNA. The ApoStrand™ ELISA is based on the sensitivity ofDNA in apoptotic cells to formamide denaturation and the detection ofthe denatured DNA with a monoclonal antibody to single-stranded DNA(ssDNA). Treatment of the liver cancer cell line HepG2 with 50 and 100μM Q10 resulted in detectable apoptosis, with a dose-response of 17% and32%, respectively (FIG. 7). These results are consistent with theobservation of Q10 inducing apoptosis in other cancer cell lines fromother tissues (e.g., SCC, SKMEL-28, MCF-7, and PC-3).

Example 4 Proteomic Analysis of Cells Treated with Q10

Cell pellets of samples treated with Q10 were analyzed using proteomicmethods. The cell pellets were lysed and treated for use in 2-D gel andWestern blot analysis. Three cell types (SKMEL-28, SCC, and nFib) weretreated with Q10 and submitted to proteomic characterization by 2-D gelelectrophoresis.

Proteomic Analysis of SKMEL-28 Cells Treated with Q10

The first experimental set processed and evaluated by Western blot and2-D gel electrophoresis was the skin cancer cell line SKMEL-28. Thisexperimental set involved SK-MEL-28 cells treated at 3, 6, 12, and 24hours with 0, 50 or 100 μM Q10.

The set of Q10 treated SK-MEL-28 samples were subjected to 2-D gelelectrophoreses (FIG. 8) and were analyzed to identify protein-levelchanges relative to the control samples. A comparative analysis of 943spots across all twenty-four gels was performed, comparing the controlsample against all of the treated samples. The analysis included theidentification of spot changes over the time course due to increase,decrease, or post-translational modification.

The analysis found thirty-two statistically significant differentialspot changes. From this, twenty non-redundant spots were excised andsubmitted for protein identification by trypsin digestion and massspectrometry characterization. The characterized peptides were searchedagainst protein databases with Mascot and MSRAT software analysis toidentify the protein (Table 2).

TABLE 2 Proteins identified to have a differential response to Q10treatment in SKMEL-28 cell. Q10 Conc. 2D Time (hr) (uM) Spot #Expression Difference Protein Name Type 3 50 528 down 1.234 cathepsin DCTSD peptidase 3 50 702 down 1.575 chaperonin containing CCT3 otherTCP1, subunit 3 3 50 74 down 1.383 eukaryotic translation EIF3Gtranslation initiation factor 3 regulator 3 50 829 down 1.074 Ribosomalprotein P2 RPLP2 other 3 50 368 down 1.121 transaldolase 1 TALDO1 enzyme6 50 452 up −1.464 eukaryotic translation EIF6 translation initiationfactor 6 regulator 6 50 175 up −1.32 Stomatin; HSPC322 STOM other 6 50827 up −1.457 Tyrosine 3/Tryptophan 5- YWHAZ enzyme monooxygenaseactivation protein 6 50 139 up −1.628 Vimentin VIM other 6 50 218 up−1.416 Vimentin VIM other 6 50 218 up −1.212 Vimentin VIM other 6 50 139up −1.036 Vimentin VIM other 6 50 507 down 1.379 Lamin B1 LMNB1 other 650 571 down 1.832 mitochandrial import TOMM22 transporter receptor Tom2212 50 166 up −1.171 ALG-2 interacting protein 1 PDCD6IP other 12 50 550up −1.747 peptidylprolyl isomerase A PPIA enzyme 12 50 613 down 1.802galectin-1 LGALS1 other 12 50 242 down 1.373 Phosphoglycerate mutase;PGAM2 phosphatase Posphomannomutase 2 24 50 326 down 1.385 glycyl-tRNAsynthase GARS enzyme 24 50 419 down 1.451 Mago-nashi homolog MAGOH other3 100 528 down −1.036 cathepsin D CTSD peptidase 3 100 702 down 1.151chaperonin containing CCT3 other TCP1, subunit 3 3 100 74 down 1.122eukaryotic translation EIF3G translation initiation factor 3 regulator 3100 829 down 1.145 Ribosomal protein P2 RPLP2 other 3 100 368 down 1.209transaldolase 1 TALDO1 enzyme 6 100 139 up −1.829 Vimentin VIM other 6100 218 up −1.761 Vimentin VIM other 6 100 452 down 1.134 eukaryotictranslation EIF6 translation initiation factor 6 regulator 6 100 252down 1.4 Sec 13 protein, Keratin II ? 6 100 827 down 1.12 Tyrosine3/Tryptophan 5- YWHAZ enzyme monooxygenase activation protein 12 100 76up −1.679 galeclin-1; keratin II LGALS1 other

A key finding in this experiment was the decrease of Transaldolase 1,which supports the premise that Q10 acts by altering the metabolic statewithin the cancer cell. Transaldolase 1 is an enzyme in the pentosephosphate pathway (also known as the hexose monophosphate shunt).Transaldolase (EC:2.2.1.2) catalyses the reversible transfer of athree-carbon ketol unit from sedoheptulose 7-phosphate to glyceraldehyde3-phosphate to form erythrose 4-phosphate and fructose 6-phosphate. Thisenzyme, together with transketolase, provides a link between theglycolytic and pentose-phosphate pathways. This is relevant tonucleotide and NADPH synthesis, to facilitate production of reducingequivalents for biosynthetic reactions and maintenance of a reducingenvironment.

A recent publication (Basta, P., et. al. August 2008, Cancer DetectPrevention, 32, 200-208) provided evidence of genetic polymorphism inTransaldolase and was linked to squamous cell carcinoma of the head andneck. Another recent publication (Qian, Y., et. al. May 2008, Biochem J,415, 123-134) identified transaldolase deficiency as a modulator ofmitochondrial homoeostasis, Ca²⁺ fluxing and apoptosis.

From these initial results, the other proteins identified by 2-D gelelectrophoresis as being modulated by Q10 in SK-MEL-28 were analyzed forknown relationships (FIG. 9). A functional evaluation of these proteinsrevealed that there was a group involved in 14-3-3-mediated signaling(PDCP61P, YWHAZ, and VIM), along with individual proteins linked to avariety of processes [cell cycle; pentose phosphate pathway (TALDOI);ceramide signaling (CTSD); aminoacyl-tRNA biosynthesis (GARS), andmitochondrial protein import (TOM22)].

Proteomic Analysis of SCC Cells Treated with Q10

Another skin cancer cell line, Squamous Cell Carcinoma (SCC), was alsoprepared and analyzed by 2-D gel electrophoreses as a follow-upexperiment the previous SK-MEL-28 analysis The SCC cells were treatedwith 100 μM Q10 for 6 hour or 24 hours before harvesting. A control ofuntreated cells was also harvested. The cell pellets were lysed and thesamples were subjected to 2-D electrophoresis (in duplicate). Analysisof over six hundred protein spots in the comparative study wasperformed, comparing the control sample against the six hour andtwenty-four hour treatments.

The top twenty-five statistically significant differential spot changeswere evaluated from the comparative analysis of the 2-D electrophoresisgels. From this, twelve spots were excised and submitted foridentification by trypsin digestion and mass spectrometrycharacterization (results summarized in Table 3 below).

TABLE 3 Proteins identified to have a differential response to 100 μMQ10 treatment in SCC cells at 6 and 24 hours. Cellular Response (foldSpot # Protein Name localization Function change) 331 Transaldolase 1TALDO1 Cytoplasm Enzyme Decrease (1.5) at 6 and 14 hr 23 Human BSCvC20ORF3 Plasma strictosidine Decrease (2.1) (chromosome 20 membranesynthase at 6 and 24 hr reading frame 3) 54 NM23 protein NME1 Nucleus,Kinase Increase (−1.2) (mitochondria?) at 6 hr, decrease at 24 hr 116two Human ESTs HSP70 Decrease (2.6) from MCF7 breast at 6 hr, furthercancer cell line decrease at 24 hr (HSP 70) 176 Heat shock 27 kDa HSPB1Cytoplasm Response to Increase (−1.9) protein 1 environmental at 6 and24 hr stresses 135 Keratin I KRT1 Cytoplasm intermediate Decrease (2.3)filaments at 6 and 24 hr 50 Keratin 14 KRT14 Cytoplasm intermediateIncrease (−1.6) filaments at 6 and 24 hr 68 Keratin 13 KRT13 Cytoplasmintermediate Increase (−1.5) filaments at 6 and 24 hr 49 Proteasome Beta7 PSMB7 Cytoplasm Proteasome Decrease (1.6) subunit at 24 hr only 93Proteasome PSME3 Cytoplasm peptidase Decrease (1.3) activator subunit 3at 24 hr only 66 Rho GDP ARHGDIA Cytoplasm Inhibitor Decrease (1.5)dissociation at 6 hr only inhibitor (GDI) alpha 1 Unknown? Decrease(9.5)

Transaldolase 1: As previously observed in the SKMEL-28 cells treatedwith Q10, the enzyme Transaldolase 1 was modulated with a decrease inlevels. This provides an independent confirmation of the previouslyobservation of a linkage between Q10 and alterations in transaldolase(and thus the metabolic state of the cell).

Transaldolase is an enzyme in the non-oxidative phase of the pentosephosphate pathway (FIG. 10). The pentose phosphate pathway is criticalin the metabolic state of cells for the generation of nicotinamideadenine dinucleotide phosphate (reduced NADH), for reductivebiosynthesis, and in the formation of ribose which is an essentialcomponent of ATP, DNA, and RNA. Transaldolase also links the pentosephosphate pathway to glycolysis. Glycolysis is the metabolic pathway bywhich cancer cells obtain the energy needed for cell survival, as themitochondrial process of oxidative phosphorylation is not utilized. Q10is an essential coenzyme factor required for oxidatative phosphorylationand mitochondrial ATP production.

BSCv: Spot 23 was a novel human protein from Chromosome 20 named BSCv.BSCv protein is also known as Adipocyte plasma membrane-associatedprotein (Gene names: APMAP or C20orf3) and is predicted to be asingle-pass type II membrane protein with sequence similarity to thestrictosidine synthase family of proteins. Q10 treatment caused areduction in the levels of this protein. This protein is not wellcharacterized, nor has its homology with strictosidine synthases beenconfirmed. Interestingly, this protein has been associated with a rolein adipocyte differentiation (Albrektsen et al., 2001). Recent proteomicstudies of human omental adipose tissue identified BSCv as one of nineproteins with differential expression for polcystic ovary syndrome(PCOS) from morbidly obese women (Corton, 2008 Hum. Reprod. 23:651-661). As a cell surface protein that responds to Q10, an antibodyagainst BSCv would be useful as a biomarker. Based on the currentresults and the literature available, BSCv may a have a potential rolein cancer and diabetes.

NM23A: Non-metastatic cells 1, protein (NM23A, also known as NME1) isthought to be a metastasis suppressor. This gene (NME1) was identifiedbecause of its reduced mRNA transcript levels in highly metastaticcells. The protein has activity as a nucleoside diphosphate kinase (NDK)and exists as a hexamer composed of ‘A’ (encoded by this gene) and ‘B’(encoded by NME2) isoforms. Mutations in this gene have been identifiedin aggressive neuroblastomas. NDK activities maintain an equilibriumbetween the concentrations of different nucleoside triphosphates suchas, for example, when GTP produced in the citric acid (Krebs) cycle isconverted to ATP. The NDK complex is associated with p53 throughinteraction with STRAP. It is noteworthy that STRAP is linked to HNF4A.Thus, NM23A is a potential protein involved in pathways important forcell control and disease treatment.

Rho GDP dissociation inhibitor (GDI) alpha: GDI Regulates the GDP/GTPexchange reaction of the Rho proteins by inhibiting the dissociation ofGDP from them, and the subsequent binding of GTP to them. The protein isupregulated in cancer cells.

Example 5 Mitochondrial Enrichment Analysis

Several lines of evidence suggested that a closer evaluation of the roleof mitochondrial proteins and cancer biology and Q10 response waswarranted. First, there is the essential role of Q10 in themitochondrial oxidative phosphorylation process for energy production innormal cells. However, the metabolic shift that occurs in cancer cellsis to energy production through the alternative pathway of glycolysis,which does not require Q10. Second, the apoptotic response of cellsrequires mitochondrial proteins to occur. Q10 has been established asstimulating apoptosis in cancer cells (Bcl-2 family proteins, cytochromec). Finally, new mitochondrial proteins were identified as beingmodulated by Q10 treatment, as exemplified by the modulation in proteinlevels of the mitochondrial import receptor protein TOM22 (seeexperiments described herein).

Production of Mitochondrial Enriched Samples

The skin cancer SKMEL-28 cells were treated with 100 μM Q10 or a mockvehicle for 6, 19, or 48 hours. The cells were harvested by washing andscraping the cells from T-160 flasks (4 for each time point). The cellswere collected by centrifugation and the pellets flash frozen and storedat −80° C. The cell pellets were resuspended and ruptured using a 2 mLDounce homogenizer. The reagents and method were obtained from aMitochondria Isolation Kit for Cultured Cells (MitoSciences, Cat#MS852). The resultant mitochondria samples were divided into 75 μLaliquots (4-5 aliquots per sample) and stored at −80° C.

Proteomic Analysis of Mitochondria Enriched Samples Isolated fromSK-MEL-28 Cells Treated with Q102-D gel electrophoresis was performed on proteins solubilized from twoaliquots of the SK-MEL-28 mitochondria enriched samples treated with 100μM Q10 for 6, 19, and 48 hours (along with the corresponding mockvehicle controls). The samples were subjected to 2-D electrophoresis (induplicate). Analysis of 525 protein spots in the comparative study wasperformed, comparing the control samples against the other time pointsamples (FIG. 11).

The nine statistically significant differential spot changes wereselected from the comparative analysis of the 2-D electrophoresis gels.From these, 9 spots were excised and submitted for identification bytrypsin digestion and mass spectrometry characterization

TABLE 4 Proteins identified to have a differential response to Q10treatment in SKMEL-28 mitochondria. Response (fold Spot # Protein NameFunction change) 11 Unknown ? ? Up (1.3) at 6 hr, drop protein to lowlevels after this 131 Unknown, same ? ? Down (1.3) at 6 hr, as spot #11,drops more for 19 and modified 48 hr 279 acyl-CoA ACOT7 Cleaves fattyacyl- Down (1.3) at 6 hr, thioesterase 7 CoA's into free fatty back tonormal at 48 hr isoform acids and CoA hBACHb 372 Pyruvate kinase PKM2catalyzes the Up (1.5) at 6 hr, back production of to normal at 48 hrphosphoenolpyruvate from pyruvate and ATP 110 ER60 protein PDIA3 Proteindisulfide Up at 19 and 48 hr isomerase 185 Keratin 10 KRT10 intermediatefilament Up only at 19 hr 202 Beta-Actin Structural protein Up only at19 hr 246 Malectin MLEC carbohydrate-binding Up only at 19 hr protein ofthe endoplasmic reticulum and a candidate player in the early steps ofprotein N- glycosylation 75 Coiled-coil CCDC58 Conserved Up at 48 hrdomain hypothetical protein - containing 58 nuclear pore forming

Acyl-CoA thioesterase 7: Acyl-CoA thioesterase 7 (ACOT7) is a member ofthe enzyme family that catalyzes the hydrolysis of fatty acyl-CoA tofree fatty acid and CoA. This enzyme thus has a role in the regulationof lipid metabolism and cellular signaling. ACOT7 has a preference forlong-chain acyl-CoA substrates with fatty acid chains of 8-16 carbonatoms (C8-C16). The exact cellular function is ACOT7 is not fullyunderstood. The transcription of this gene is activated by sterolregulatory element-binding protein 2, thus suggesting a function incholesterol metabolism.

The results in this Example indicate that ACOT7 is potentially involvedin the metabolism of Q10, either directly or indirectly. Thus, targetingACOT7 could facilitate modulation of intercellular levels of Q10 andthus impact cellular Q10 effects.

Pyruvate kinase: Pyruvate kinase is an enzyme involved in the last stepof glycolysis. It catalyzes the transfer of a phosphate group fromphosphoenolpyruvate (PEP) to ADP, yielding one molecule of pyruvate andone molecule of ATP.

The protein is presumably that of PKM2, the type 2 isoform, as this wasidentified from the mitochondria enriched SK-MEL-28 sample. This isoformis well known to be involved in tumor cell formation and regulation.

Quantification of Q10 Levels in Mitochondria

A method for the simultaneous determination of Coenzyme Q10, (Q10) andthe reduced form ubiquinol-10 (Q10H2) was implemented based upon arecently published method (Ruiz-Jimenez, 2007, J. Chroma A, 1175,242-248) through the use of LC-MS-MS with electrospray ionization (ESI)in the positive mode. The highly selective identification and sensitivequantitation of both Q10 and Q10H2 is possible, along with theidentification of other selected lipids. An aliquot of the mitochondrialenriched samples from SK-MEL-28 treated with 100 μM Q10 were subject toa conventional pre-treatment based on protein precipitation,liquid-liquid extraction, evaporation to dryness and reconstitution with95:5 methanol/hexane (v/v).

In this analysis, Q10, Q10H2, and Q9 were quantitated (Table 5). Thelevels of the related molecule Q9 were low, and near the level ofdetection. The level of the untreated samples were relativelyconsistent, with the 6 hour Q10 treated sample having this same level.To control for sample variance in total material, the levels ofcholesterol was also measured to confirm that the differences were notdue to sample size errors. When the Q10 levels were corrected againsttotal protein values obtained by protein extraction other aliquots ofthe same mitochondrial preps, the relative ratios were comparative.Thus, a significant increase in Q10 levels was obtained at 19 hours(˜3-fold) with an even larger increase by the 48 hour time point(˜6-fold) (FIG. 12).

TABLE 5 HPLC-MS Quantification results for the levels of Q10 present inmitochondrial enriched samples from SK-MEL-28 cells treated with 100 μMQ10 in the media. Peak Area ng/Sample μg/sample File Sample Injection Q9Q10 Q9 Q10 Q10H₂ Cholesterol 081204-05 100 ng Std 245,342 352792081204-06 6 hr mock#1 10% 2560 32649 1.04 9.25 081204-07 Solvent Blank#15 ul 3781 3174 1.54 0.9 081204-08 Solvent Blank#2 5 ul 2396 4399 0.981.25 081204-09 6 hr mock#2 20% 1572 36328 0.64 10.3 081204-10 SolventBlank#3 10 ul  1722 2504 0.7 0.71 081204-11 48 hr Q10 treated 20% 4879164496 1.99 46.63 0.28 13.86 081204-12 48 hr mock 20% 2412 25552 0.987.24 0.09 13.04 081204-13 6 hr Q10 treated 20% 692 25427 0.28 7.21081204-14 19 hr Q10 treated 20% 1161 59164 0.47 16.77 081204-15 19 hrmock 20% 901 19999 0.37 5.67

A surprising result from this study was the finding that the Q10 wassupplied to the cells as the oxidized form. For the 48 hour samples, thereduced form Q10H2 was also measured and found to be present insignificantly lower amounts (0.28 ng/sample of CoQ10H2 as compared to46.63 ng/sample of CoQ10). There was a general increase (3-fold) in thelevels of Q10H2 in the Q10 treated 48 hour sample, although the levelswere near the presumed detection limit of the assay. Interestingly, theoxidized form (Q10) can act as a pro-oxidant in biological systems.According to the literature, when human plasma was evaluated for Q10 andQ10H2, the majority (90%) of the molecule was found in the reduced formof Q10H2 (Ruiz-Jimenez, 2007, J. Chroma A, 1175, 242-248) which can actas an anti-oxidant.

Thus, these results confirm and quantitate that the levels of Q10increase in the mitochondria upon the exogenous addition of Q10 to themedia. A surprising and unexpected discovery was that Q10 was maintainedin the supplied oxidized form (pro-oxidant) and not converted to thereduced (anti-oxidant) form of Q10H2 in any significant amounts.

Example 6 Real-Time PCR Arrays Experiment 1: Apoptosis Array

As discussed above in Example 3, exposure of cancer cells to Q10 inducesa portion of these cells to die due to apoptotic processes. To identifyproteins that were involved in the Q10 response, real-time polymerasechain reaction (RT-PCR) methods were employed to identify changes in thelevel of mRNA for genes/proteins involved in targeted pathway arrays forapoptosis.

Using PCR arrays as a screening tool, a spectrum of molecular targetsthat would potentially offer an insight to the mode of biological actionof Q10 within the cells were thus evaluated. Changes in mRNA levels wereevaluated using real-time PCR quantification to assess mRNA levels inpre-selected subsets containing 80 pathway specific targets.

For the interpretation of mRNA results, the genes that were altered intheir mRNA transcription by a two-fold level were identified andevaluated. The level of gene transcription to produce mRNA only providesa rough estimate of potential changes, in the level of the expressedprotein. The skilled artisan will appreciate that each mRNA may havedifferent rates at which it is degraded or its translationinefficiently, thus resulting in differing amounts of protein.

SkBr-3 Cells Treated with 50 um Q10 for 24 Hours

The assay method of RT-PCR was utilized to provide a measure of mRNAlevel changes to a total of 84 apoptotic pathway related proteins. Theexperiments with the real-time PCR apoptosis analysis on SkBr3 with Q10(24 hr) identified the following mRNA's being affected: Bcl2, Bcl2L1,Bcl2L11, Birc6, Bax, Xiap, Hprt1, Apaf1, Abl1, Braf. These results againprovided supporting evidence for the apoptotic response of cancer cellsto Q10 treatment.

TABLE 6A Up-Down Symbol Regulation Unigene Refseq Description GnameBCL2L1 13.1957 Hs.516966 NM_138578 BCL2-like 1 BCL-XL/S BNIP2 6.3291Hs.646490 NM_004330 BCL2/adenovirus E1B BNIP-2/NIP2 19 kDa interactingprotein 2 BCL2 5.4717 Hs.150749 NM_000633 B-cell CLL/lymphoma 2 Bcl-2BIRC6 4.7966 Hs.150107 NM_016252 Baculoviral IAP APOLLON/Brepeat-containing 6 RUCE (apollon) BCL2L11 4.6012 Hs.469658 NM_006538BCL2-like 11 (apoptosis BAM/BIM facilitator) XIAP 4.3832 Hs.356076NM_001167 X-linked inhibitor of API3/BIRC4 apoptosis BRAF 4.3832Hs.550061 NM_004333 V-raf murine sarcoma B-raf viral oncogene homolog1/BRAF1 B1 BAX 3.896 Hs.631546 NM_004324 BCL2-associated X Bax zetaprotein APAF1 2.6244 Hs.708112 NM_001160 Apoptotic peptidase CED4/DKFZpactivating factor 1 781B1145 HPRT1 −160.6748 Hs.412707 NM_000194Hypoxanthine HGPRT/HPRT phosphoribosyltransferase 1 (Lesch-Nyhansyndrome)

Results that are consistent from three independent experiments fromSK-MEL-28 cells are summarized below in Table 6B. Many genes areregulated in SCC cells as well with 100 μM Q10 treatment. The genes inthe Apoptosis array that appear to be regulated in SCC cells aredescribed in Table 7. We find that many genes are regulated at 6 hours,both in SK-MEL-28 cells and in SCC cells. By 24 hours, the regulation isdecreased. Genes that appear to be regulated in both SK-MEL-28 cells andin SCC cells are described in Table 8.

TABLE 6B Genes in SK-MEL-28 cells regulated by 100 μM Q10 treatment whenanalyzed by the Apoptosis Array. Symbol Description Regulation LocationPossible Functions ABL1 C-abl oncogene 1, Down Regulated NucleusTyrosine Kinase receptor tyrosine at 72 hours kinase BAG1BCL2-associated Up Regulated at Cytoplasm Anti-apoptotic, athanogene 48hours glucocorticoid receptor pathway BCL2 B-cell Down RegulatedCytoplasm Cell death CLL/lymphoma 2 at 48 hours BCL2A1 BCL2-related DownRegulated Cytoplasm Regulates Caspases, protein A1 at 48 hoursphosphorylates TP73 BCL2L1 BCL2-like 1 Down Regulated Cytoplasm CaspaseInhibitor at 72 hours BCL2L10 BCL2-like 10 Down Regulated CytoplasmCaspase Activator (apoptosis at 48 hours facilitator) BCL2L11 BCL2-like11 Down Regulated Cytoplasm Pro-Apoptotic, (apoptosis at 48 hoursCaspase3 Activator facilitator) BIRC3 Baculoviral IAP Down RegulatedCytoplasm Anti-apoptotic repeat-containing 3 at 6 hours BIRC8Baculoviral IAP Down Regulated Cytoplasm Activates Caspaserepeat-containing 8 at 48 hours CARD8 Caspase recruitment Down RegulatedNucleus Caspase Activator domain family, at 48 hours member 8 CASP14Caspase 14, Down Regulated Cytoplasm Apoptosis related apoptosis-relatedat 48 hours cysteine peptidase cysteine peptidase CASP5 Caspase 5, DownRegulated Cytoplasm Apoptosis related apoptosis-related at 48 hourscysteine peptidase cysteine peptidase CD40LG CD40 ligand (TNF DownRegulated Extracellular CD40 receptor superfamily, at 48 hours Spacebinding member 5, hyper- IgM syndrome) CIDEA Cell death-inducing UpRegulated at Cytoplasm Pro-Apoptotic DFFA-like effector a 48 hours FADDFas (TNFRSF6)- Down Regulated Cytoplasm Pro-Apoptotic associated viadeath at 6 hours domain FAS Fas (TNF receptor Up Regulated at PlasmaPro-Apoptotic superfamily, 48 hours Membrane member 6) FASLG Fas ligand(TNF Down Regulated Extracellular Pro-Apoptotic superfamily, at 48 hoursSpace member 6) GADD45A Growth arrest and Up Regulated at Nucleus GrowthArrest DNA-damage- 48 hours inducible, alpha HRK Harakiri, BCL2 DownRegulated Cytoplasm Pro-Apoptotic interacting protein at 48 hours(contains only BH3 domain) PYCARD PYD and CARD Down Regulated CytoplasmApoptotic Protease domain containing at 6 hours Activator TNF Tumornecrosis Up Regulated at Extracellular TNF receptor factor (TNF 48 hoursthen Space binding superfamily, down regulated member 2) TNFRSF10A Tumornecrosis Up Regulated at Plasma Caspase Activator factor receptor 48hours then Membrane superfamily, down regulated member 10a TNFRSF10BTumor necrosis Down Regulated Plasma p53 signaling, factor receptor at72 hours Membrane caspase activation. superfamily, member 10b TNFRSF1ATumor necrosis Down Regulated Plasma Pro-apoptotic factor receptor at 72hours Membrane superfamily, member 1A TNFRSF21 Tumor necrosis DownRegulated Plasma Activates Caspase factor receptor at 48 hours Membranesuperfamily, member 21 CD27 CD27 molecule Down Regulated Plasma CaspaseInhibitor at 48 hours Membrane TNFRSF9 Tumor necrosis Down RegulatedPlasma Pro-apoptotic factor receptor at 48 hours Membrane superfamily,member 9 TNFSF10 Tumor necrosis Upregulated at ExtracellularPro-apoptotic factor (ligand) 48 hours Space superfamily, member 10 TP73Tumor protein p73 Down Regulated Nucleus Transcription factor at 48hours TRAF3 TNF receptor- Down Regulated Cytoplasm Zinc-finger domainassociated factor 3 at 48 hours TRAF4 TNF receptor- Down RegulatedCytoplasm Zinc-finger domain associated factor 4 at 48 hours

TABLE 7 Genes in SCC cells that are regulated by 100 μM Q10 treatmentwhen analyzed by the Apoptosis Array. Symbol Description Regulation.AKT1 V-akt murine thymoma viral oncogene Down regulated at 6 hours andhomolog 1 then up regulated at 24 hours. BAG4 BCL2-associated athanogene4 Up regulated at 24 hours. BAX BCL2-associated X protein Up regulatedat 24 hours. BCL2 B-cell CLL/lymphoma 2 Up regulated at 24 hours. BCL2L1BCL2-like 1 Down regulated at 6 hours and then up regulated at 24 hours.BIRC3 Baculoviral IAP repeat-containing 3 Down regulated at 6 hours.BNIP3 BCL2/adenovirus E1B 19 kDa interacting Down regulated at 24 hours.protein 3 CARD6 Caspase recruitment domain family, Down regulated at 6hours. member 6 CASP6 Caspase 6, apoptosis-related cysteine Up regulatedat 24 hours. peptidase CASP7 Caspase 7, apoptosis-related cysteine Upregulated at 24 hours. peptidase CD40 CD40 molecule, TNF receptor Downregulated at 6 hours. superfamily member 5 FADD Fas (TNFRSF6)-associatedvia death Up regulated at 24 hours. domain GADD45A Growth arrest andDNA-damage- Up regulated at 24 hours. inducible, alpha HRK Harakiri,BCL2 interacting protein Up regulated at 24 hours. (contains only BH3domain) TNFRSF21 Tumor necrosis factor receptor Down regulated at 6hours. superfamily, member 21 TNFRSF25 Tumor necrosis factor receptorDown regulated at 6 hours and superfamily, member 25 then up regulatedat 24 hours. CD27 CD27 molecule Down regulated at 6 hours. TNFRSF9 Tumornecrosis factor receptor Down regulated at 6 hours. superfamily, member9 TNFSF10 Tumor necrosis factor (ligand) Up regulated at 24 hours.superfamily, member 10 CD70 CD70 molecule Down regulated at 6 hours.TP53 Tumor protein p53 Up regulated at 24 hours. TP73 Tumor protein p73Down regulated at 6 hours and then up regulated at 24 hours. TRAF2 TNFreceptor-associated factor 2 Up regulated at 24 hours.

TABLE 8 Genes from the apoptosis array regulated with 100 μM Q10treatment in both SK-MEL-28 and SCC cells. Symbol Description BCL2B-cell CLL/lymphoma 2 BCL2L1 BCL2-like 1 (Bcl-xl) BIRC3 Baculoviral IAPrepeat-containing 3 FADD Fas (TNFRSF6)-associated via death domainGADD45A Growth arrest and DNA-damage-inducible, alpha TNFRSF21 Tumornecrosis factor receptor superfamily, member 21 CD27 CD27 moleculeTNFRSF9 Tumor necrosis factor receptor superfamily, member 9 TNFSF10Tumor necrosis factor (ligand) superfamily, member 10 TP73 Tumor proteinp73 TRAF2 TNF receptor-associated factor 2

Interestingly, the altered mRNA levels showed a significantup-regulation in a series of apoptitic proteins, with Bcl-xl one of thehighest. This was also observed in the protein array experiments onSK-MEL-28 cells.

Bcl-xl is a transmembrane molecule in the mitochondria (Bcl-xl standsfor “Basal cell lymphoma-extra large”). It is involved in the signaltransduction pathway of the FAS-L and is one of several anti-apoptoticproteins which are members of the Bcl-2 family of proteins. It has beenimplicated in the survival of cancer cells. However, it is known thatalternative splicing of human Bcl-x mRNA may result in at least twodistinct Bcl-x mRNA species, Bcl-xL and Bcl-xS. The predominant proteinproduct (233 amino acids) is the larger Bcl-x mRNA, Bcl-xL, whichinhibits cell death upon growth factor withdrawal (Boise et al., 1993.Cell 74, 597-608). Bcl-xS, on the other hand, inhibits the ability ofBcl-2 to inhibit cell death and renders cells more susceptible toapoptotic cell death. The employed assays utilized do not distinguishwhich isoform of Bcl-x is being upregulated. The Bcl-x isoform beingupregulated by CoQ10 in these studies may be determined by routinemethods known in the art, e.g., by using RT-PCR methods to evaluate theratio of the two mRNA splicing isoforms (Bcl-xL vs Bcl-sL).

From the survey of apoptotic related proteins it was observed multiplepro- and anti-apoptotic factors were in the BCL-2 family or thatinteract with these factors have modulated expression levels (BCL2L11,BNIP2, BAG1, HRK, BAK1, BCL2, BCL2L1). These proteins governmitochondrial outer membrane permeabilization.

An early marker for apoptotic response is observed with the upregulationof Caspase-9 (16 hour) which is consistent with previous observations ofapoptosis with caspase 3/7 proteins. Induction of stress signalingpathways causes release of cytochrome c from mitochondria and activationof apaf-1 (apoptosome), which in turn cleaves the pro-enzyme ofcaspase-9 into the active form. Once intiated caspase-9 goes on tocleave procaspase-3 & procaspase-7 to trigger additional apoptoticpathways.

There is also a consistent linkage to the tumor necrosis factor receptorfamily of proteins being modulated.

A strong down regulation of tumor protein p73 is also noted. Analyses ofmany tumors typically found in humans including breast and ovariancancer show a high expression of p73 when compared to normal tissues incorresponding areas. Recent finding are suggesting that deregulated overexpression of transcription factors within the body involved in cellcycle regulation and synthesis of DNA in mammalian cells (i.e.: E2F-1),induces the expression of p73. The suggestion is that p73 may be anoncoprotein, but may involve different mechanism that the related p53protein. A schematic showing mapping of the apoptosis pathway isprovided in FIG. 13.

SKMEL-28 Cells

From the survey of apoptotic related proteins it was observed multiplepro- and anti-apoptotic factors were in the BCL-2 family or thatinteract with these factors have modulated expression levels (BCL2L11,BNIP2, BAG1, HRK, BAK1, BCL2, BCL2L1). These proteins governmitochondrial outer membrane permeabilization.

An early marker for apoptotic response is observed with the upregulationof Caspase-9 (16 hour) which is consistent with previous observations ofapoptosis with caspase 3/7 proteins. Induction of stress signalingpathways causes release of cytochrome c from mitochondria and activationof apaf-1 (apoptosome), which in turn cleaves the pro-enzyme ofcaspase-9 into the active form. Once intiated caspase-9 goes on tocleave procaspase-3 & procaspase-7 to trigger additional apoptoticpathways.

TABLE 9 Changes in mRNA levels for SKMEL-28 cells treated with 100 μMA10, evaluated by RT-PCR arrays focused around apoptotic pathways 6 hr16 hr 24 hr 72 hr Refseq Description Symbol Q10 Q10 Q10 Q10 NM_006538BCL2-like 11 BCL2L11 2.13 2.41 1.92 2.51 (apoptosis facilitator)NM_000875 Insulin-like IGF1R 1.77 1.09 1.33 1.25 growth factor 1receptor NM_004048 Beta-2- B2M 1.74 1.76 1.58 3.11 microglobulinNM_003921 B-cell BCL10 1.55 1.87 1.48 −3.11 CLL/lymphoma 10 NM_004330BCL2/adenovirus BNIP2 1.46 1.51 1.57 −1.61 E1B 19 kDa interactingprotein 2 NM_005157 C-abl oncogene 1, ABL1 1.42 2.77 −1.22 −2.03receptor tyrosine kinase NM_004323 BCL2-associated BAG1 1.41 1.44 −1.61−2.45 athanogene NM_001229 Caspase 9, CASP9 1.32 3.96 1.83 1.14apoptosis-related cysteine peptidase NM_003806 Harakiri, BCL2 HRK 1.184.52 2.73 −1.14 interacting protein (contains only BH3 domain) NM_001924Growth arrest and GADD45A 1.07 3.34 1.13 −2.36 DNA-damage- inducible,alpha NM_001188 BCL2-antagonist/ BAK1 1.06 2.73 −1.00 −4.54 killer 1NM_004295 TNF receptor- TRAF4 −1.91 2.63 −1.58 −740.66 associated factor4 NM_003842 Tumor necrosis TNFRSF10B −2.07 1.53 −1.81 −710.49 factorreceptor superfamily, member 10b NM_000633 B-cell BCL2 −2.98 −1.63 −2.82−11.36 CLL/lymphoma 2 NM_001242 CD27 molecule CD27 −3.40 −2.38 −1.35−12.72 NM_014430 Cell death- CIDEB −3.48 1.56 −3.69 −2.59 inducing DFFA-like effector b NM_001065 Tumor necrosis TNFRSF1A −4.53 2.28 −3.30 1.22factor receptor superfamily, member 1A NM_005427 Tumor protein TP73−4.66 −9.80 −8.71 −26.96 p73 NM_003844 Tumor necrosis TNFRSF10A −4.84−5.26 −4.33 −11.84 factor receptor superfamily, member 10a NM_138578BCL2-like 1 BCL2L1 −4.94 −1.80 −6.17 −7.04 NM_001165 Baculoviral IAPBIRC3 −13.68 −1.98 −2.42 −3.42 repeat-containing 3

There is a consistent linkage to the tumor necrosis factor receptorfamily of proteins being modulated.

A strong down regulation of tumor protein p73 is also noted. Analyses ofmany tumors typically found in humans including breast and ovariancancer show a high expression of p73 when compared to normal tissues incorresponding areas. Recent finding are suggesting that deregulated overexpression of transcription factors within the body involved in cellcycle regulation and synthesis of DNA in mammalian cells (i.e.: E2F-1),induces the expression of p73. The suggestion is that p73 may be anoncoprotein, but may involve different mechanism that the related p53protein.

Experiment 2: Real-Time PCR Arrays Using Oxidative Stress andAntioxidant Defense Array

To identify proteins that were involved in the Q10 response, real-timepolymerase chain reaction (RT-PCR) methods were employed to identifychanges in the level of mRNA's for genes/proteins involved in targetedpathway arrays for oxidative stress and antioxidant defense.

Table 10 below lists the genes that are regulated in SK-MEL28 cells with100 μM Q10 treatment. Results are given only for those genes that areregulated in two independent experiments. Although there is asignificant amount of gene regulation seen at 6 hours, most significantchanges in RNA levels are seen at 48 hours.

TABLE 10 Genes in SK-MEL-28 cells that are regulated by 100 μM Q10treatement as seen in the Oxidative Stress and Antioxidant DefenseArrays. Symbol Description Regulation Location Possible Functions. ALBAlbumin Down Regulation at Extracellular Carrier protein, anti- 48 hoursspace apoptotic AOX1 Aldehyde oxidase 1 Up regulation from 16 CytoplasmProduces free radicals, drug hours metabolic process. APOEApolipoprotein E Down Regulation at Extracellular Lipid metabolism 48hours space ATOX1 ATX1 antioxidant Down Regulation at Cytoplasm Coppermetabolism protein 1 homolog 48 hours (yeast) BNIP3 BCL2/adenovirus DownRegulation at Cytoplasm Anti-apoptotic E1B 19 kDa 48 hours interactingprotein 3 CSDE1 Cold shock domain Down Regulation at CytoplasmTranscriptional regulation. containing E1, 48 hours RNA-binding CYBACytochrome b-245, Down Regulation at Cytoplasm Apoptotic, alphapolypeptide 48 hours CYGB Cytoglobin Down Regulation at CytoplasmPeroxidase, Transporter. 48 hours DHCR24 24- Down Regulation at 6Cytoplasm Electron carrier, binds to dehydrocholesterol hours TP53,involved in apoptosis. reductase DUOX1 Dual oxidase 1 Up Regulation at48 Plasma Calcium ion binding, hours Membrane electron carrier. DUOX2Dual oxidase 2 Down Regulation at Unknown Calcium ion binding. 48 hoursEPHX2 Epoxide hydrolase Down Regulation at Cytoplasm Arachidonic acide2, cytoplasmic 48 hours metabolism. EPX Eosinophil Down Regulation atCytoplasm Phenyl alanine metabolism, peroxidase 48 hours apoptosis. GPX2Glutathione Down Regulation at Cytoplasm Electron carrier, binds toperoxidase 2 48 hours TP53, involved in apoptosis. (gastrointestinal)GPX3 Glutathione Up Regulation at 48 Extracellular Arachidonic acidperoxidase 3 hours space metabolims, up regulated in (plasma)carcinomas. GPX5 Glutathione Up Regulation at 48 ExtracellularArachidonic acid peroxidase 5 hours space metabolism. (epididymalandrogen-related protein) GPX6 Glutathione Down Regulation atExtracellular Arachidonic acid peroxidase 6 48 hours space metabolism.(olfactory) GSR Glutathione Down Regulation at Cytoplasm Glutamate andglutathione reductase 48 hours metabolism, apoptosis. GTF2I General DownRegulation at 6 Nucleus Transcriptional activator, transcription factorhours transcription of fos. II, i KRT1 Keratin 1 Up Regulation at 48Cytoplasm Sugar Binding. (epidermolytic hours hyperkeratosis) LPOLactoperoxidase Down Regulation at Extracellular Phenyl alaninemetabolism. 48 hours space MBL2 Mannose-binding Down Regulation atExtracellular Complement signaling, lectin (protein C) 2, 48 hours spacepattern recognition in soluble (opsonic receptors. defect) MGST3Microsomal Upregulation at 16 Cytoplasm Xenobiotic metabolism.glutathione S- hours transferase 3 MPO Myeloperoxidase Down Regulationat Cytoplasm Anti-apoptotic, phenyl 48 hours alanine metabolism. MPV17MpV17 Down Regulation at 6 Cytoplasm Maintenance of mitochondrial innerhours mitochondrial DNA. membrane protein MT3 Metallothionein 3 DownRegulation at Cytoplasm Copper ion binding. 48 hours NCF1 Neutrophilcytosolic Down Regulation Cyoplasm Produces free radicals. factor 1,(chronic from 6 hours granulomatous disease, autosomal 1) NCF2Neutrophil cytosolic Up Regulation at 48 Cytoplasm Electron carrier.factor 2 (65 kDa, hours chronic granulomatous disease, autosomal 2) NME5Non-metastatic cells Down Regulation at Unknown Kinase, Purine and 5,protein expressed 48 hours pyrimidine metabolism. in (nucleoside-diphosphate kinase) NOS2A Nitric oxide Down Regulation at CytoplasmGlucocorticoid receptor synthase 2A 48 hours signaling, apoptosis.(inducible, hepatocytes) OXR1 Oxidation resistance 1 Down Regulation atCytoplasm Responds to oxidative stress. 48 hours PDLIM1 PDZ and LIM UpRegulation at 48 Cytoplasm Transcriptional activator. domain 1 (elfin)hours PIP3-E Phosphoinositide- Down Regulation at Cytoplasm Peroxidase.binding protein 48 hours PIP3-E PRDX2 Peroxiredoxin 2 Down Regulation at6 Cytoplasm Role in phenyl alanine hours metabolism. Role in cell death.PRDX4 Peroxiredoxin 4 Down Regulation Cytoplasm Thioredoxin peroxidase.from 24 hours PREX1 Phosphatidylinositol Down Regulation at CytoplasmForms oxygen free radicals. 3,4,5-trisphosphate- 48 hours dependent RACexchanger 1 PRG3 Proteoglycan 3 Down Regulation at Extracellular Role incell death. 48 hours space PTGS1 Prostaglandin- Down Regulation atCytoplasm arachidonic acid endoperoxide 48 hours metabolism,prostaglandin synthase 1 synthesis. (prostaglandin G/H synthase andcyclooxygenase) PTGS2 Prostaglandin- Up Regulation at 48 Cytoplasmarachidonic acid endoperoxide hours metabolism, prostaglandin synthase 2synthesis. (prostaglandin G/H synthase and cyclooxygenase) PXDNPeroxidasin Up Regulation at 48 Unknown binds to TRAF4, calcium homologhours ion binding, iron ion (Drosophila) binding. PXDNL Peroxidasin DownRegulation at Unknown peroxidase, calcium ion homolog 48 hours binding,iron ion binding. (Drosophila)-like RNF7 Ring finger protein 7 UpRegulation at 16 Nucleus apoptotic, copper ion hours binding, ubiquitinpathway. SGK2 Serum/glucocorticoid Down Regulation at Cytoplasm Kinase,potasium channel regulated kinase 2 48 hours regulator. SIRT2 Sirtuin(silent Up regulation at 16 Nucleus Transcription factor. mating typehours information regulation 2 homolog) 2 (S. cerevisiae) SOD1Superoxide Up Regulation at 16 Cytoplasm Apoptotic, Caspase dismutase 1,soluble hours Activator. (amyotrophic lateral sclerosis 1 (adult)) SOD2Superoxide Up regulation at 16 Cytoplasm Apoptotic, Regulated bydismutase 2, hours TNF. mitochondrial SOD3 Superoxide Down Regulation atExtracellular Pro-apoptotic dismutase 3, 48 hours space extracellularSRXN1 Sulfiredoxin 1 Down Regulation at Cytoplasm DNA binding, homolog(S. cerevisiae) 48 hours oxidoreductase TPO Thyroid peroxidase DownRegulation at Plasma iodination of thyroglobulin, 48 hours Membranetyrosine metabolism, phenylalanine metabolism. TTN Titin Down Regulationat Cytoplasm Actin cytoskeleton 48 hours signaling, integrin signalingTXNDC2 Thioredoxin Down Regulation at Cytoplasm Pyrimidine metabolismdomain-containing 2 48 hours (spermatozoa)

The Neutrophil cytosolic factor 2 (NCF2, 65 kDa, chronic granulomatousdisease, autosomal 2) was one of the initial top induced mRNA's(observed at 6 hours). Subsequently at the 16 hour time point andonward, Neutrophil cytosolic factor 1 (NCF1) (chronic granulomatousdisease, autosomal 1) was induced at very high levels after an initiallag phase.

Neutrophil cytosolic factor 2 is the cytosolic subunit of themulti-protein complex known as NADPH oxidase commonly found inneutrophils. This oxidase produces a burst of superoxide which isdelivered to the lumen of the neutrophil phagosome.

The NADPH oxidase (nicotinamide adenine dinucleotide phosphate-oxidase)is a membrane-bound enzyme complex. It can be found in the plasmamembrane as well as in the membrane of phagosome. It is made up of sixsubunits. These subunits are:

-   -   a Rho guanosine triphosphatase (GTPase), usually Rac1 or Rac2        (Rac stands for Rho-related C3 botulinum toxin substrate)

Five “phox” units. (Phox stands for phagocytic oxidase.)

-   -   P91-PHOX (contains heme)    -   p22phox    -   p40phox    -   p47phox (NCF1)    -   p67phox (NCF2)

It is noted that another NADPH oxidase levels do not change. The enzymeis NOX5, which is a novel NADPH oxidase that generates superoxide andfunctions as a H+ channel in a Ca(2+)-dependent manner

In addtition Phosphatidylinositol 3,4,5-trisphosphate-dependent RACexchanger 1 (PREX1) was also upregulated. This protein acts as a guaninenucleotide exchange factor for the RHO family of small GTP-bindingproteins (RACs). It has been shown to bind to and activate RAC1 byexchanging bound GDP for free GTP. The encoded protein, which is foundmainly in the cytoplasm, is activated byphosphatidylinosito1-3,4,5-trisphosphate and the beta-gamma subunits ofheterotrimeric G proteins.

The second major early induced protein was Nitric oxide synthase 2A(inducible, hepatocytes) (NOS2A). Nitric oxide is a reactive freeradical which acts as a biologic mediator in several processes,including neurotransmission and antimicrobial and antitumoralactivities. This gene encodes a nitric oxide synthase which is expressedin liver and is inducible by a combination of lipopolysaccharide andcertain cytokines.

Superoxide dismutase 2, mitochondrial (SOD2) is a member of theiron/manganese superoxide dismutase family. It encodes a mitochondrialprotein that forms a homotetramer and binds one manganese ion persubunit. This protein binds to the superoxide byproducts of oxidativephosphorylation and converts them to hydrogen peroxide and diatomicoxygen. Mutations in this gene have been associated with idiopathiccardiomyopathy (IDC), premature aging, sporadic motor neuron disease,and cancer.

An example of a down regulated protein is Forkhead box M1 (FOXM1), whichis known to play a key role in cell cycle progression where endogenousFOXM1 expression peaks at S and G2/M phases. Recent studies have shownthat FOXM1, regulates expression of a large array of G2/M-specificgenes, such as Plk1, cyclin B2, Nek2 and CENPF, and plays an importantrole in maintenance of chromosomal segregation and genomic stability.The FOXM1 gene is now known as a human proto-oncogene. Abnormalupregulation of FOXM1 is involved in the oncogenesis of basal cellcarcinoma (BCC). FOXM1 upregulation was subsequently found in themajority of solid human cancers including liver, breast, lung, prostate,cervix of uterus, colon, pancreas, and brain. Further studies with BCCand Q10 should evaluate FOXM1 levels.

SKMEL-28 Cells

Further experiments were carried out using SKMEL-28 cells. The level ofmRNA present in SKMEL-28 cells treated with 100 μM Q10 were compared tothe levels in untreated cells at various time points using real-time PCRmethods (RT-PCR). The PCR array (SABiosciences) is a set of optimizedreal-time PCR primer assays on 96-well plates for pathway or diseasefocused genes as well as appropriate RNA quality controls. The PCR arrayperforms gene expression analysis with real-time PCR sensitivity and themulti-gene profiling capability of a microarray.

TABLE 11 Listing and classification of mRNA levels evaluated in theOxidative Stress and Antioxidant Defense PCR Array. After six hours oftreatment with 100 μM Q10 on SKMEL-28 cells, the largest changes to themRNA levels are indicated by highlighting the protein code(increased-bold; decreased-underlined; or no change-grey).

TABLE 12 Time course evaluation of 100 μM treatment of SKMEL-28. ThemRNA level changes were monitored by RT-PCR methods and oxidative stressand antioxidant defense proteins array was evaluated. 6 hr 16 hr 24 hr48 hr 72 hr Refseq Symbol Description Q10 Q10 Q10 Q10 Q10 NM_000265 NCF1Neutrophil cytosolic 0 high 3.3829 15.7838 31.5369 factor 1, (chronicgranulomatous disease, autosomal 1) NM_012423 RPL13A Ribosomal protein−0.9025 3.1857 2.5492 4.9253 7.82 L13a NM_020820 PREX1Phosphatidylinositol −3.2971 2.867 0.3222 6.3719 7.4763,4,5-trisphosphate- dependent RAC exchanger 1 NM_012237 SIRT2 Sirtuin(silent mating −0.9025 4.0829 4.4766 5.7166 6.6257 type informationregulation 2 homolog) 2 (S. cerevisiae) NM_005125 CCS Copper chaperonefor −0.6206 3.0077 3.452 2.9801 6.1539 superoxide dismutase NM_181652PRDX5 Peroxiredoxin 5 −2.995 3.0454 3.5381 4.7955 6.0169 NM_016276 SGK2Serum/glucocorticoid 0 0 0 0.5995 5.937 regulated kinase 2 NM_003551NME5 Non-metastatic cells 5, −0.6652 3.1138 3.3694 3.1549 5.782 proteinexpressed in (nucleoside-diphosphate kinase) NM_004417 DUSP1 Dualspecificity −0.6998 0.5902 2.7713 3.321 5.5375 phosphatase 1 NM_001752CAT Catalase −0.8589 2.8424 0.1046 3.8557 5.3988 NM_000041 APOEApolipoprotein E −0.8212 3.2069 −0.9543 3.7694 5.3315 NM_000101 CYBACytochrome b-245, −0.3945 4.3475 3.9208 6.2452 5.0762 alpha polypeptideNM_000433 NCF2 Neutrophil cytosolic 1.2266 3.0077 0.0954 5.476 0 factor2 (65 kDa, chronic granulomatous disease, autosomal 2) NM_000963 PTGS2Prostaglandin-endoperoxide −0.6912 2.7046 2.6552 4.0553 −3.3022 synthase2 (prostaglandin G/H synthase and cyclooxygenase) NM_183079 PRNP Prionprotein (p27-30) −0.2144 3.5236 2.9086 5.0837 −3.9396 (Creutzfeldt-Jakobdisease, Gerstmann Strausler-Scheinker syndrome, fatal familialinsomnia) NM_004052 BNIP3 BCL2/adenovirus E1B −2.9376 3.3288 4.312−18.2069 −4.8424 19 kDa interacting protein 3 NM_000242 MBL2Mannose-binding lectin −0.3622 −1.9072 −3.0142 −1.1854 −6.4544 (proteinC) 2, soluble (opsonic defect) NM_021953 FOXM1 Forkhead box M1 −0.81350.068 −0.9216 3.3655 −10.0953

The Neutrophil cytosolic factor 2 (NCF2, 65 kDa, chronic granulomatousdisease, autosomal 2) was one of the initial top induced mRNA's(observed at 6 hours). Subsequently at the 16 hour time point andonward, Neutrophil cytosolic factor 1 (NCF1) (chronic granulomatousdisease, autosomal 1) was induced at very high levels after an initiallag phase.

Neutrophil cytosolic factor 2 is the cytosolic subunit of themulti-protein complex known as NADPH oxidase commonly found inneutrophils. This oxidase produces a burst of superoxide which isdelivered to the lumen of the neutrophil phagosome. The NADPH oxidase(nicotinamide adenine dinucleotide phosphate-oxidase) is amembrane-bound enzyme complex. It can be found in the plasma membrane aswell as in the membrane of phagosome. It is made up of six subunits.These subunits are:

-   -   a Rho guanosine triphosphatase (GTPase), usually Rac1 or Rac2        (Rac stands for Rho-related C3 botulinum toxin substrate)    -   Five “phox” (phagocytic oxidase) units.        -   P91-PHOX (contains heme)        -   p22phox        -   p40phox        -   p47phox (NCF1)        -   p67phox (NCF2)

It is noted that another NADPH oxidase levels do not change. The enzymeis NOX5, which is a novel NADPH oxidase that generates superoxide andfunctions as a H+ channel in a Ca(2+)-dependent manner

In addition Phosphatidylinositol 3,4,5-trisphosphate-dependent RACexchanger 1 (PREX1) was also upregulated. This protein acts as a guaninenucleotide exchange factor for the RHO family of small GTP-bindingproteins (RACs). It has been shown to bind to and activate RAC1 byexchanging bound GDP for free GTP. The encoded protein, which is foundmainly in the cytoplasm, is activated byphosphatidylinosito1-3,4,5-trisphosphate and the beta-gamma subunits ofheterotrimeric G proteins.

The second major early induced protein was Nitric oxide synthase 2A(inducible, hepatocytes) (NOS2A). Nitric oxide is a reactive freeradical which acts as a biologic mediator in several processes,including neurotransmission and antimicrobial and antitumoralactivities. This gene encodes a nitric oxide synthase which is expressedin liver and is inducible by a combination of lipopolysaccharide andcertain cytokines.

An example of a down regulated protein is FOXM1, which is known to playa key role in cell cycle progression where endogenous FOXM1 expressionpeaks at S and G2/M phases. Recent studies have shown that FOXM1,regulates expression of a large array of G2/M-specific genes, such asPlk1, cyclin B2, Nek2 and CENPF, and plays an important role inmaintenance of chromosomal segregation and genomic stability. The FOXM1gene is now known as a human proto-oncogene. Abnormal upregulation ofFOXM1 is involved in the oncogenesis of basal cell carcinoma (BCC).FOXM1 upregulation was subsequently found in the majority of solid humancancers including liver, breast, lung, prostate, cervix, uterus, colon,pancreas, and brain.

Experiment 3: Real-Time PCR Arrays Using Heat Shock Array

Heat Shock Arrays were run for SCC cells and the data of regulated genesis summarized below in Table 13.

TABLE 13 Genes from the Heat Shock Protein array regulated with 100 μMQ10 treatment in SCC cells. Symbol Description Regulation. Location.Possible functions. CCT6B Chaperonin Down Cytoplasm Protein folding andcontaining TCP1, regulated at 24 protein complex subunit 6B (zeta hoursassembly. 2) DNAJA1 DnaJ (Hsp40) Up regulated Nucleus Responds to DNAhomolog, at 6 hours. damage and changes in protein subfamily A, folding.member 1 DNAJB13 DnaJ (Hsp40) Down Unknown Protein folding and related,subfamily regulated at 6 apoptosis. B, member 13 hours. DNAJB5 DnaJ(Hsp40) Down Unknown Binds to HSP, homolog, regulated at 6 involved inprotein subfamily B, hours. folding and in protein member 5 complexassembly. DNAJC12 DnaJ (Hsp40) Down Unknown Binds to HSP, homolog,regulated at 6 involved in protein subfamily C, hours. folding and inprotein member 12 complex assembly. DNAJC4 DnaJ (Hsp40) Down CytoplasmBinds to HSP, homolog, regulated at 6 involved in protein subfamily C,hours. folding and in protein member 4 complex assembly. DNAJC5B DnaJ(Hsp40) Down Unknown Involved in protein homolog, regulated at 6 foldingresponds to subfamily C, hours. changes in protein member 5 betafolding. HSPA8 Heat shock Up regulated Cytoplasm Regulates TNF, binds 70kDa protein 8 at 6 hours. BAG1, STUB1, TP53, involved in apoptosis.HSPH1 Heat shock Up regulated Cytoplasm Binds to HSPA8, 105 kDa/110 kDaat 6 hours. important for protein protein 1 folding, responds to proteinunfolding and stress.

Experiment 4: Real-Time PCR Arrays Using Diabetes Array

The experiments described in this example were performed to test theoverall hypothesis that Q10 would have an impact on multiple genes andalter the metabolic state of a cell. The mRNA from SKMEL-28 cellstreated with 100 μM Q10 was evaluated by RT-PCR against a panel oftarget proteins involved in diabetes and related pathways. Results fromthis experiment demonstrate that several proteins involved in glycolyicpathways and insulin processing are altered in their mRNA expressionlevels (summarized in Table 14).

TABLE 14 Major mRNA level changes to SKMEL-28 cells treated with 100 μMQ10 for 16 hours. Fold Change after 16 hours (100 μM Refseq DescriptionSymbol Q10) NM_000162 Glucokinase (hexokinase 4) GCK 8.5386 NM_178849Hepatocyte nuclear factor 4, HNF4A 8.421 alpha NM_005249 Forkhead box G1FOXG1 4.6396 NM_000599 Insulin-like growth factor IGFBP5 2.2721 bindingprotein 5 NM_001101 Actin, beta ACTB −2.0936 NM_002863 Phosphorylase,glycogen; PYGL −2.65 liver (Hers disease, glycogen storage disease typeVI) NM_001065 Tumor necrosis factor TNFRSF1A −2.8011 receptorsuperfamily, member 1A NM_021158 Tribbles homolog 3 TRIB3 −2.8011(Drosophila) NM_003749 Insulin receptor substrate 2 IRS2 −2.9404NM_004578 RAB4A, member RAS RAB4A −3.1296 oncogene family NM_004176Sterol regulatory element SREBF1 −3.5455 binding transcription factor 1NM_004969 Insulin-degrading enzyme IDE −4.4878 NM_005026Phosphoinositide-3-kinase, PIK3CD −6.8971 catalytic, delta polypeptideNM_000208 Insulin receptor INSR −8.6099 NM_003376 Vascular endothelialgrowth VEGFA −15.5194 factor A NM_001315 Mitogen-activated proteinMAPK14 −74.3366 kinase 14

The results of this initial experiment show that the mRNA levels for avariety of insulin related proteins were modulated in both directions.The results indicate that Q10 would have an impact on diabetic diseasetreatment and/or evaluation.

Further experiments were next conducted to confirm the results aboveobtained from SK-MEL-28 cells treated with Q10. Many of the genes inSK-MEL-28 cells are regulated as early as 6 hours after Q10 treatment.However, the initial regulation becomes less evident by 16 and 24 hours.Around 48 hours, we find that many of the genes in the Diabetes arrayare again strongly regulated. Results that are consistent from two ormore or independent experiments are summarized below in Table 15. SCCcells also appeared to exhibit regulation in some genes, both at 6 and24 hours after Q10 treatment. These results from SCC cells aresummarized in Table 16 while genes that are regulated both in SK-MEL-28cells and in SCC cells are summarized in Table 17.

TABLE 15 Genes in SK-MEL-28 cells regulated by 100 μM Q10 treatment whenanalyzed by the Diabetes Array. Symbol Description Regulation. LocationPossible Function ADRB3 Adrenergic, beta-3-, Down Regulated Plasma cAMPsignaling, receptor at 48 hours membrane G-protein signaling CEACAM1Carcinoembryonic Down Regulated Extracellular Anti-apoptotic,antigen-related cell at 48 hours space positive regulation adhesionmolecule 1 of angiogenesis. (biliary glycoprotein) CEBPA CCAAT/enhancerbinding Up regulated at 48 Nucleus Glucocorticoid protein (C/EBP), alphahours receptor signaling, VDR/RXR activation. CTLA4 CytotoxicT-lymphocyte- Down Regulated Plasma T cell receptor associated protein 4at 48 hours Membrane signaling, activates CASP8. DUSP4 Dual specificityDown Regulated Nucleus Phosphatase phosphatase 4 at 48 hours ENPP1Ectonucleotide Down Regulated Plasma Negative regulatorpyrophosphatase/phospho at 48 hours membrane of the insulin diesterase 1receptor pathway FOXC2 Forkhead box C2 (MFH-1, Down Regulated NucleusAnti-apoptotic, mesenchyme forkhead 1) at 48 hours transcription factorG6PD Glucose-6-phosphate Up regulated at 48 Cytoplasm Pentose Phosphatedehydrogenase hours, then down Pathway, regulated Glutathionemetabolism. HMOX1 Heme oxygenase Down Regulated Cytoplasm Heme oxygenase(decycling) 1 at 48 hours decycling ICAM1 Intercellular adhesion DownRegulated Plasma Regulated by molecule 1 (CD54), at 48 hours membraneatorvastatin, human rhinovirus receptor processes some caspases. IL4RInterleukin 4 receptor Down Regulated Plasma Up regulation by at 48hours membrane TP73, binds to IRS1 and IRS2 IRS1 Insulin receptorsubstrate 1 Up regulated at 48 Plasma Binds Insulin hours then downmembrane receptor regulated IRS2 Insulin receptor substrate 2 DownRegulated Plasma IGF-1 signaling at 48 hours membrane NSFN-ethylmaleimide- Down Regulated Cytoplasm GABA signaling sensitivefactor at 48 hours PIK3CD Phosphoinositide-3- Down Regulated CytoplasmKinase kinase, catalytic, delta at 48 hours polypeptide PPARG Peroxisomeproliferator- Down Regulated Nucleus Transcriptional activated receptorgamma at 48 hours factor PRKCB1 Protein kinase C, beta 1 Down RegulatedCytoplasm PKC family at 48 hours SELL Selectin L (lymphocyte DownRegulated Plasma Activates RAS, adhesion molecule 1) at 48 hoursmembrane MAPK SREBF1 Sterol regulatory element Up regulated at 48Nucleus Transcriptional binding transcription hours then down factorfactor 1 regulated STXBP1 Syntaxin binding protein 1 Down RegulatedCytoplasm Present in myelin at 48 hours enriched fraction. TGFB1Transforming growth Up regulated at 48 Extracellular Pro-apoptoticfactor, beta 1 hours then down space regulated NKX2-1 NK2 homeobox 1Down Regulated Nucleus Transcriptional at 48 hours activator TNF Tumornecrosis factor Up regulated at 48 Extracellular Pro-apoptotic (TNFsuperfamily, hours space member 2) TNFRSF1A Tumor necrosis factor DownRegulated Plasma Pro-apoptotic receptor superfamily, at 72 hoursmembrane member 1A VEGFA Vascular endothelial Up regulated at 58Cytoplasm Kinase growth factor A hours then down regulated

TABLE 16 Genes in SCC cells regulated by 100 μM Q10 treatment whenanalyzed by the Diabetes Array. Symbol Description Regulation. G6PDGlucose-6-phosphate dehydrogenase Down regulated at 6 hours. ICAM1Intercellular adhesion molecule 1 Down regulated at 6 (CD54), humanrhinovirus hours. receptor INPPL1 Inositol polyphosphate Down regulatedat 6 phosphatase-like 1 hours. NOS3 Nitric oxide synthase 3 (endothelialDown regulated at 6 cell) hours. PIK3CD Phosphoinositide-3-kinase,catalytic, Down regulated at 6 delta polypeptide hours. PPARA Peroxisomeproliferative activated Down regulated at 6 receptor, alpha hours. PYGLPhosphorylase, glycogen; liver Down regulated at 6 (Hers disease,glycogen hours. storage disease type VI) SREBF1 Sterol regulatoryelement binding Down regulated at 6 transcription factor 1 hours. STXBP2Syntaxin binding protein 2 Down regulated at 6 hours. TNF Tumor necrosisfactor (TNF Down regulated at 6 superfamily, member 2) hours. TNFRSF1ATumor necrosis factor receptor Down regulated at 6 superfamily, member1A and 24 hours. VEGFA Vascular endothelial growth factor A Downregulated at 6 hours.

TABLE 17 Genes from the diabetes array regulated with 100 μM Q10treatment for both SK-MEL-28 and SCC cells. Symbol Description. G6PDGlucose-6-phosphate dehydrogenase ICAM1 Intercellular adhesion molecule1 (CD54), human rhinovirus receptor PIK3CD Phosphoinositide-3-kinase,catalytic, delta polypeptide SREBF1 Sterol regulatory element bindingtranscription factor 1 TNF Tumor necrosis factor (TNF superfamily,member 2) TNFRSF1A Tumor necrosis factor receptor superfamily, member 1AVEGFA Vascular endothelial growth factor A

The mRNA levels for a variety of insulin related proteins were modulatedin both directions. Q10 has an impact on regulation of cellularmetabolism, and thus influences metabolic disregluation diseases such asdiabetes. Two proteins that were significantly modulated are furtherdiscussed below.

Mitogen-activated protein kinase 14 (MAPK14): Mitogen-activated proteinkinase 14 (MAPK14) is a member of the MAP kinase family. MAP kinases actas an integration point for multiple biochemical signals, and areinvolved in a wide variety of cellular processes such as proliferation,differentiation, transcription regulation and development. Results fromthis experiment show that the MAPK14 was significantly down-regulated.

Hepatocyte nuclear factor 4, alpha (HNF4A): HNF4 (Hepatocyte NuclearFactor 4) is a nuclear receptor protein mostly expressed in the liver,gut, kidney, and pancreatic beta cells that is critical for liverdevelopment. In humans, there are two isoforms of NHF4, alpha and gammaencoded by two separate genes HNF4A and HNF4G respectively. (See, e.g.,Chartier F L, Bossu J P, Laudet V, Fruchart J C, Laine B (1994).“Cloning and sequencing of cDNAs encoding the human hepatocyte nuclearfactor 4 indicate the presence of two isoforms in human liver”. Gene 147(2): 269-72.)

HNF4 was originally classified as an orphan receptor. However HNF4 wasfound later to be constitutively active by virtue of being continuouslybound to a variety of fatty acids. (See, e.g., Sladek F (2002).“Desperately seeking . . . something”. Mol Cell 10 (2): 219-221 and JumpD B, Botolin D, Wang Y, Xu J, Christian B, Demeure O (2005). “Fatty acidregulation of hepatic gene transcription”. J Nutr 135 (11)). The ligandbinding domain of HNF4, as with other nuclear receptors, adopts acanonical alpha helical sandwich fold (see, e.g., Wisely G B, Miller AB, Davis R G, Thornquest A D Jr, Johnson R, Spitzer T, Sefler A, ShearerB, Moore J T, Miller A B, Willson T M, Williams S P (2002). “Hepatocytenuclear factor 4 is a transcription factor that constitutively bindsfatty acids”. Structure 10 (9): 1225-34 and Dhe-Paganon S, Duda K,Iwamoto M, Chi Y I, Shoelson S E (2002). “Crystal structure of the HNF4alpha ligand binding domain in complex with endogenous fatty acidligand”. J Biol Chem 277 (41): 37973-6) and interacts with co-activatorproteins. (See, e.g., Duda K, Chi Y I, Shoelson S E (2004). “Structuralbasis for HNF-4-alpha activation by ligand and coactivator binding”. JBiol Chem 279 (22): 23311-6).

Mutations in the HNF4-α gene have been linked to maturity onset diabetesof the young (MODY). (See, e.g., Fajans S S, Bell G I, Polonsky K S(2001). “Molecular mechanisms and clinical pathophysiology ofmaturity-onset diabetes of the young”. N Engl J Med 345 (13): 971-80.)

Hepatocyte nuclear factor 4 (HNF4) is a tissue-specific transcriptionfactor known to regulate a large number of genes in hepatocytes andpancreatic cells. Although HNF4 is highly expressed in some sections ofthe kidney, little is known about its role in this organ and aboutHNF4-regulated genes in the kidney cells. The abundance and activity ofHNF4 are frequently reduced in renal cell carcinoma (RCC) indicatingsome tumor suppressing function of HNF4 in renal cells. Interestingly,many of the genes regulated by HNF4 have been shown to be deregulated inRCC microarray studies. These genes (ACY1, WT1, SELENBP1, COBL, EFHD1,AGXT2L1, ALDH5A1, THEM2, ABCB1, FLJ14146, CSPG2, TRIM9 and HEY1) aregood candidates for genes whose activity is changed upon the decrease ofHNF4 in RCC.

In the structure of the ligand binding domain of HNF4alpha (1M7W.pdb;Dhe-Paganon (2002) JBC, 277, 37973); a small lipid was observed andwhich co-purified from E. coli production. The crystal contains twoconformations of the protein, where the elongated helix 10 and shorthelix 12 have alternate conformations. Upon examination of the lipidbinding region, it was interesting to observe that there are two exitsregions. One exit region holds the small lipids head group, and it isnoted that several pocket regions are co-localized with this exit port.A hypothesis would be that Q10 binds specifically to this transcriptionfactor. When Q10 in modeled into this lipid binding tunnel, the Q10 ringwould fit into the surface pocket (FIG. 28). A known loss-of-functionmutation (E276Q) would have the potential to order the residues liningthis surface pocket, and thus have a negative impact on the putative Q10binding.

In addition, with this Q10 binding model, the hydrophobic tail wouldextend out of the internal cavity and would then interact with theelongated helix 10. Thus, this interaction could potential alter theconformation of the helix 10/12 group. This may then alter theactivation/inactivation equilibrium of the transcription factoractivity.

Example 7 Antibody MicroArray Analysis

The evaluation of protein concentration due to the presence of Q10 wasevaluated through the utilization of antibody microarray methods. Themicroarray contained antibodies for over 700 proteins, sampling a broadrange of protein types and potential pathway markers.

An initial experiment to assess changes at the protein concentrationlevel in cells treated with Q10 was conducted with an antibodymicroarray (Panorama XP725 Antibody Array, Sigma) and SK-MEL-28 cellstreated for 6 or 24 hour. The cells were harvested and extracted toobtain a soluble protein supernatant. Two portions of protein (˜1 mgtotal) from each sample (at 1 mg/mL) were each label with fluorescentdye (Cy3 and Cy5, respectively). The excess dye was removed from theprotein and the material utilized for the microarray incubations. Tocompare two time point samples, equal amounts of protein were mixed,with each sample being of the different label type (e.g., 3 hour extractlabeled with Cy3 was mixed with the 24 hour extract labeled with Cy5).After incubation with the microarray chip (according to manufacturesrecommended protocols), the chips were washed and dried. The microarrayswere scanned with a fluorescent laser scanner to measure the relativefluorescence intensity of the Cy3 and Cy5 dyes.

TABLE 18 Proteins with increased levels in SK-MEL-28 cells after 24 hourtreatment with 50 μM Q10 Name Ratio Cdk1 0.1 DcR1 0.1 Protein Kinase Cb20.1 Tumor Necrosis Factor 0.1 Soluble Receptor II BAD 0.1 Caspase13 0.2FBI1 PAKEMON 0.2 Zyxin 0.2 Cdc25A 0.3 PIASx 0.3 Nerve Growth Factor b0.3 Protein Tyrosine 0.3 Phosphatase PEST hBRM hSNF2a 0.4 GRP94 0.4Calmodulin 0.4 Serine Threonine Protein 0.4 Phosphatase 2C a b ARC 0.4NeurabinII 0.4 Nitric Oxide Synthase 0.4 bNOS Serine Threonine Protein0.4 Phosphatase 1b Heat Shock Protein 110 0.4 Serine Threonine Protein0.4 Phosphatase 1g1 COX II 0.5 HSP70 0.5 BLK 0.5 Cytokeratin 8 12 0.5BUBR1 0.5 FOXC2 0.5 Serine Threonine Protein 0.5 Phosphatase 2 A Bg MSH60.5 DR6 0.5 Rad17 0.5 BAF57 0.5 Transforming Growth 0.5 Factorb pan BTK0.5 SerineThreonine Protein 0.5 Phosphatase 2 A/B pan2 CNPase 0.5 SynCAM0.5 Proliferating Cell Nuclear 0.5 Antigen

TABLE 19 Proteins with increased levels in SK-MEL-28 cells after 24 hourtreatment with 50 μM Q10 Name Ratio BclxL 4.2 BID 3.7 Bmf 3.7 PUMA bbc33.0 Zip Kinase 2.8 Bmf 2.8 DcR2 2.7 E2F1 2.7 FAK pTyr577 2.5 FKHRL1FOXO3a 2.5 MTBP 2.5 Connexin 32 2.5 Annexin VII 2.4 p63 2.4 SUMO1 2.4IAfadin 2.3 MDMX 2.3 Pyk2 2.3 RIP Receptor Interacting 2.3 Protein RICK2.3 IKKa 2.3 Bclx 2.3 Afadin 2.2 Proliferating Cell Protein 2.2 Ki67Histone H3 pSer28 2.2 CASK LIN2 2.2 Centrin 2.2 TOM22 2.1 Nitric OxideSynthase 2.1 Endothelial eNOS Protein Kinase Ba 2.1 Laminin 2.1 MyosinIb Nuclear 2.1 Caspase 7 2.1 MAP Kinase 2 ERK2 2.1 KIF17 2.1 Claspin 2.1GRP75 2.1 Caspase 6 2.1 ILP2 2.1 aActinin 2.1 Vitronectin 2.1 DRAK1 2.1PTEN 2.1 Grb2 2.1 HDAC4 2.0 HDAC7 2.0 Nitric Oxide Synthase bNOS 2.0HDAC2 2.0 p38 MAPK 2.0 Reelin 2.0 Protein Kinase Cd 2.0 cerbB3 2.0 hSNF5INI1 2.0 Protein Kinase Ca 2.0 Glutamate receptor NMDAR 2.0 2a Leptin2.0 Dimethyl Histone H3 2.0 diMeLys4 BID 2.0 MeCP2 2.0 Nerve growthfactor receptor 2.0 p75 Myosin Light Chain Kinase 2.0 cRaf pSer621 2.0GRP78 BiP 2.0 cMyc 2.0 Raf1 2.0 MTA2 MTA1L 2.0 Sir2 2.0 ATF2 pThr69 712.0 Protein Kinase C 2.0 Protein Kinase Cb2 2.0

In order to confirm the previously observed apoptosis proteins, and toexpand the evaluation into a larger number of pro-apoptosis andanti-apoptosis proteins, two assay methods were chosen which werecapable of screening the broad family of proteins potentially involved.

First, an antibody micro array (Panorama XP725 Antibody Array, Sigma)was utilized to screen over 700 protein antibodies to assess changes atthe protein concentration level in SK-MEL-28 cells treated for 24 hourswith 50 μM Q10.

From the Antibody array experiments, on SKMEL-28 with Q10 (24 hr), thefollowing are some of the identified proteins with altered levels:Bcl-xl, Bmf, BTK, BLK, cJun (pSer63), Connexin 32, PUMA bbc3, BID, Par4,cCbl. The key conclusion from this initial study was that the expectedpro-apoptosis proteins are altered.

Antibody Microarray for SK-MEL-28

An antibody micro array (Panorama XP725 Antibody Array, Sigma) wasutilized to screen over 700 protein antibodies to assess changes at theprotein concentration level in SK-MEL-28 cells treated for 24 hours with50 μM Q10.

TABLE 20 Changes in protein levels in SKMEL-28 treated with 50 μM Q10SKMEL28 HEKa Antibody Q10/ SKMEL28/ Q10/ Number SKMEL28 HEKa HEKa Name(Sigma) control control control BclxL B9429 2.46 1.04 1.83 PUMA bbc3P4743 2.31 1.14 2.14 Bmf B1559 2.23 1.12 2.11 Bmf B1684 2.09 1.13 1.74cJun pSer63 J2128 1.99 1.14 1.85 BLK B8928 1.94 1.05 1.51

From the Antibody array experiments, on SKMEL-28 with Q10 (24 hr), thefollowing are some of the identified proteins with altered levels:Bcl-xl, Bmf, BTK, BLK, cJun (pSer63), Connexin 32, PUMA bbc3, BID, Par4,cCb1. These data confirm that the levels of pro-apoptosis proteins arealtered upon incubation with elevated levels of exogenously added Q10.

Bcl-xl (“Basal cell lymphoma-extra large”) is a transmembrane moleculein the mitochondria. It is involved in the signal transduction pathwayof the FAS-L and is one of several anti-apoptotic proteins which aremembers of the Bcl-2 family of proteins. It has been implicated in thesurvival of cancer cells. However, it is known that alternative splicingof human Bcl-x mRNA may result in at least two distinct Bcl-x mRNAspecies, Bcl-xL and Bcl-xS. The predominant protein product (233 aminoacids) is the larger Bcl-x mRNA, Bcl-xL, which inhibits cell death upongrowth factor withdrawal (Boise et al., 1993. Cell 74, 597-608). Bcl-xS,on the other hand, inhibits the ability of Bcl-2 to inhibit cell deathand renders cells more susceptible to apoptotic cell death.

TABLE 21 Proteins with increased levels in SCC cells after hourtreatment with 100 μM Q10. Name Ratio PUMA bbc3 3.81 HDAC7 3.21 BID 3.12MTBP 3.00 p38 MAP Kinase 2.93 NonActivated PKR 2.87 TRAIL 2.86 DR5 2.86Cdk3 2.82 NCadherin 2.71 Reelin 2.68 p35 Cdk5 Regulator 2.63 HDAC10 2.60RAP1 2.59 PSF 2.56 cMyc 2.55 methyl Histone H3 2.54 MeLys9 HDAC1 2.51F1A 2.48 ROCK1 2.45 Bim 2.45 FXR2 2.44 DEDAF 2.44 DcR1 2.40 APRIL 2.40PRMT1 2.36 Pyk2 pTyr580 2.34 Vitronectin 2.33 Synaptopodin 2.32Caspase13 2.30 Syntaxin 8 2.29 DR6 2.29 BLK 2.28 ROCK2 2.28 Sir2 2.25DcR3 2.24 RbAp48 RbAp46 2.21 OGlcNAc Transferase 2.21 GRP78 BiP 2.20Sin3A 2.20 p63 2.20 Presenilin1 2.19 PML 2.18 PAK1pThr212 2.17 HDAC82.16 HDAC6 2.15 Nitric Oxide Synthase 2.15 Inducible iNOS Neurofibromin2.15 Syntaxin 6 2.13 Parkin 2.12 Rad17 2.11 Nitric Oxide Synthase 2.10bNOS TIS7 2.09 OP18 Stathmin (stathmin 2.08 1/oncoprotein 18)phospho-b-Catenin pSer45 2.07 NeurabinII 2.07 e Tubulin 2.07 PKB pThr3082.07 Ornithine Decarboxylase 2.07 P53 BP1 2.06 Pyk2 2.05 HDAC5 2.05Connexin 43 2.05 a1Syntrophin 2.04 MRP1 2.04 cerbB4 2.03 SNitrosocysteine 2.03 SGK 2.02 Rab5 2.01 Ubiquitin Cterminal 2.01Hydrolase L1 Myosin Ib Nuclear 2.00 Par4 Prostate Apoptosis 2.00Response 4

TABLE 22 Proteins with reduced levels in SCC cells after 24 hourtreatment with 100 μM Q10. Name Ratio AP1 0.68 Centrin 0.55 CUGBP1 0.67Cystatin A 0.69 Cytokeratin CK5 0.60 Fibronectin 0.63 gParvin 0.70Growth Factor Independence1 0.63 Nerve Growth Factor b 0.60 ProCaspase 80.72 Rab7 0.62 Rab9 0.73 Serine Threonine Protein Phosphatase 0.71 1g1Serine Threonine Protein Phosphatase 2 0.73 A Bg SKM1 0.70 SLIPR MAGI30.67 Spectrin a and b 0.70 Spred2 0.66 TRF1 0.74

Example 8 Western Blot Analysis

The first experiment processed and evaluated by Western blot and 2-D gelelectrophoresis was carried out on the skin cancer cell line SKMEL-28.This experimental set involved SK-MEL-28 cells treated at 3, 6, 12, and24 hours with 50 or 100 μM Q10.

A variety of cell types were evaluated by Western blot analysis againstan antibody for Bcl-xL (FIG. 14), an antibody for Vimentin (FIG. 15), aseries of antibodies for mitochondrial oxidative phosphorylationfunction (FIGS. 16-21) and against a series of antibodies related tomitochondrial membrane integrity (FIGS. 22-27). The results from theseexperiments demonstrated that several of the examined proteins wereupregulated or downregulated as a result of cell treatment with Q10.

Example 9 Diabetes Related Genes Identified as Being Modulated at themRNA Level by Treatment of Pancreatic Cancer Cells (PaCa2) with 100 umQ10

Diabetes arrays were run for samples treated with 100 uM Q10 at varioustimes after treatment. Experiments were carried out essentially asdescribed above. The various genes found to be modulated upon Q10treatment are summarized in Table 23 below. The results showed that thefollowing genes are modulated by Q10 treatment: ABCC8, ACLY, ADRB3,CCL5, CEACAM1, CEBRA, FOXG1, FOXP3, G6PD, GLP1R, GPD1, HNF4A, ICAM1,IGFBP5, INPPL1, IRS2, MAPK14, ME1, NFKB1, PARP1, PIK3C2B, PIK3CD,PPARGC1B, PRKAG2, PTPN1, PYGL, SLC2A4, SNAP25, HNF1B, TNRFSF1A, TRIB3,VAPA, VEGFA, IL4R and IL6.

TABLE 23 Genes from the diabetes array whose expression is regulatedwith 100 μM Q10 and their possible functions in a cell Up-regulated(grey) and down-regulated (white) Gene Name Gene Function. ADRB cAMPsignaling, G-protein signaling CCL5 Natural ligands for CCR5 and isregulated by TNF. CEACAM1 Anti-apoptotic, positive regulation ofangiogenesis. GLPR1 Increases Insulin and decreases glucagon secretionfrom the pancreas. GPD1 Carbohydrate metabolism, NADH oxidation. ICAM1Regulated by atorvastatin, processes some caspases. MAPK14 DNA damagecheckpoint, angiogenesis, glucose metabolic process. PARP1 DNA repair,regulates TP53, NOS2A, NFKB, telomere maintenance. PIK3C2BPhosphoinositide mediated signaling, regulates AKT and AKT1. PIK3CDKinase PYGL carbohydrate metabolism, regulates glycogen and glycogensynthase. SLC2A4 regulates glucose and is regulated by INS and insulin.SNAP25 regulation of insulin secretion, nerotransmitter uptake. CEBPAGlucocorticoid receptor signaling, VDR/RXR activation. FOXP3 RegulatesIL4, IL2. G6PD Pentose Phosphate Pathway, Glutathione metabolism. IGFBP5Regulation of cell growth, regulated by IGF1 INPPL1 Regulates Akt andglycogen. IRS2 IGF-1 signaling ME1 Regulates malic acid and is regulatedby T3. NFKB1 Regulates IL6 and TNF. PPARGC1B Regulated by MAPK14 PRKAG2Fatty acid, cholesterol biosynthesis. PTPN1 dephosphorylates JAK2 andEGFreceptor kinase. VEGFA Kinase, angiogenesis. IL4R Up regulation byTP73, binds to IRS1 and IRS2 HNF1B HNF4A TNFRSF1A Pro-apoptotic TRIB3Regulates AKT1 and negative regulator of NFkB. VAPA Regulates NFkB,vesicle trafficking.

Example 10 Angiogenesis Related Genes Identified as Being Modulated atthe mRNA Level by Treatment of Pancreatic Cancer Cells (PaCa2) with 100μM Q10

Angiogenesis arrays were run for samples treated with 100 uM Q10 atvarious times after treatment. Experiments were carried out essentiallyas described above. The various genes found to be modulated upon Q10treatment are summarized in Table 24 below. The results showed that thefollowing genes are modulated by Q10 treatment: AKT1, ANGPTL4, ANGPEP,CCL2, CDH4, CXCL1, EDG1, EFNA3, EFNB2, EGF, FGF1, ID3, IL1B, 1L8, KDR,NRP1, PECAM1, PROK2, SERPINF1, SPHK1, STAB1, TGFB1, VEGFA and VEGFB.

TABLE 24 A list of genes from the angiogenesis array whose expression isregulated with 100 μM Q10 and their possible functions in a cellUp-regulated (grey) and down-regulated (white) Gene Gene Function.ANGPTL4 antiangiogenesis, negative regulator of apoptosis, lipidmetabolism. CDH5 blood vessel maturation, cell-adhesion, negativeregulator of cell proliferation. FGF1 Cell adhesion, cell proliferation.AKT1 carbohydrate metabolic process, glycogen biosynthetic process,glucose metabolic process, insulin receptor signaling pathway,activation of pro-apoptotic gene products, apoptotic mitochondrialchanges ANPEP proteolysis, multicellular organismal development, celldifferentiation CCL2 chemotaxis, anti-apoptosis, JAK-STAT cascade, organmorphogenesis, viral genome replication CXCL1 chemotaxis, inflammatoryresponse, immune response, negative regulation of cell proliferation,actin cytoskeleton organization and biogenesis. EDG1 positive regulationof cell proliferation, transmission of nerve impulse, regulation of celladhesion, neuron differentiation, positive regulation of cell migration,positive regulation of Ras EFNB2 cell-cell signaling, regulated byVEGFA. EGF activation of MAPKK activity, positive regulation of mitosis,DNA replication ILIB response to glucocorticoid stimulus, apoptosis,signal transduction, cell-cell signaling, negative regulation of cellproliferation IL8 cell cycle arrest KDR VEGF pathway, regulated by AKT.NRP1 cell adhesion, signal transduction, cell-cell signaling, cellproliferation, regulated by VEGFA PECAM1 cell adhesion, regulated byTNF. PROK2 activation of MAPK, anti-apoptosis, cell proliferation,regulates AKT, SPHK1 anti-apoptosis, cell proliferation, regulatesmitosis, cell migration. STAB1 inflammatory response, cell adhesion,receptor-mediated endocytosis, cell-cell signaling, negative regulationof angiogenesis, defense response to bacterium VEGFA anti-apoptosis,regulates TNF, regulated by HIF1.

Example 11 Apoptosis Related Genes Identified as Being Modulated at themRNA Level by Treatment of Pancreatic Cancer Cells (PaCa2) with 100 μMQ10

Apoptosis arrays were run for samples treated with 100 uM Q10 at varioustimes after treatment. Experiments were carried out essentially asdescribed above. The various genes found to be modulated upon Q10treatment are summarized in Table 25 below. The results showed that thefollowing genes are modulated by Q10 treatment: ABL1, AKT1, Bcl2L1,BclAF1, CASP1, CASP2, CASP6, CIDEA, FADD, LTA, TNF, TNFSF10A andTNFSF10.

TABLE 25 A list of genes from the apoptosis array whose expression isregulated with 100 μM Q10 and their possible functions in a cellUp-regulated (Grey) and down-regulated (white) Gene Gene Function. CASP1Pro-Apoptotic, Regulates IL1B, regulated by TNF. CASP6 Pro-Apoptotic,regulates PARP, MCL1, APP TNF cell proliferation, differentiation,apoptosis, lipid metabolism, and coagulation TNFSF10 Pro-Apoptotic,regulates caspases. ABL1 Regulates Bcl2L1, TP53, Pro-apoptotic, actincytoskeleon organization and biogenesis. AKT1 Prop-apoptotic, apoptoticmitochondrial changes, carbohydrate transport, response to heat, glucosemetabolism, IGF signaling pathway. BclAF1 Pro-Apoptotic. Bcl2L1Anti-Apoptotic, release of cytochrome c from mitochondria, regulatesCaspases, binds to BAD, BAX, BCl2L11 CASP2 Anti-Apoptotic. CIDEAPro-Apoptotic FADD Pro-Apoptotic LTA Pro-Apoptotic TNFSF10A CaspaseActivator

Example 12 PCR Diabetes Arrays on Liver Cancer (HepG2) Cells

HepG2 (liver cancer) cells were treated with either the vehicle for 24hours or 100 μM Q10 for different times. The treatment was initiated on1×10⁵ cells per well, following the procedure utilized in the PaCa2cells (above, Examples 9-11). However, the total amount of RNA that wasextracted from these samples was lower than expected. Reversetranscription is normally done using 1 μg of total RNA (determined bymeasurement at 260 nm). The maximum volume that can be used per reversetranscription is 8 μl. Since the RNA concentration was low, the RT-PCRarray analysis using the vehicle, and Q10 treated samples from 16 hoursand 48 hours was performed using 0.44 μg of RNA. The arrays provided aninitial analysis of trends and patterns in HepG2 gene regulation with100 μM Q10 treatment, as summarized in Table 26 below. The resultsshowed that each of the genes PPARGC1A, PRKAA1 and SNAP25 weredownregulated at 16 hours following treatment (by approximately 20 fold,6 fold and 5 fold, respectively). At 48 hours following treatment,PPARGC1A and PRKAA1 had normalized or were slightly upregulated, whileSNAP25 was downregulated by approximately 2 fold.

TABLE 26 List of genes regulated in the Diabetes Arrays when HepG2 cellswere treated with 100 μM Q10 Gene Gene name Gene Function. PPARGC1Aperoxisome proliferator- Involved in cell death, activated receptorproliferation, cellular respiration gamma, coactivator 1 andtransmembrane potential. alpha PRKAA1 protein kinase, AMP- RegulatesTP53 and is involved activated, alpha 1 in apoptosis, regulatescatalytic subunit glycolysis, regulates metabolic enzyme activities.SNAP25 synaptosomal-associated Plays in transport, fusion, protein, 25kDa exocytosis and release of molecules.

Example 13 PCR Angiogenesis Array on Liver Cancer (HEPG2) Cells

HepG2 (liver cancer) cells were treated with either the vehicle for 24hours or 100 μM Q10 for different times. The treatment was initiated on1×105 cells per well, following the procedure utilized in the PaCa2cells (above Examples 9-11). However, the total amount of RNA that wasextracted from these samples was lower than expected. Reversetranscription is normally done using 1 μg of total RNA (determined bymeasurement at 260 nm). The maximum volume that can be used per reversetranscription is 8 μl. Since the RNA concentration was low, the RT-PCRarray analysis using the vehicle, and Q10 treated samples from 16 hoursand 48 hours was performed using 0.44 μg of RNA. The arrays provided aninitial analysis of trends and patterns in HepG2 gene regulation with100 μM Q10 treatment, as summarized in Table 27 below. The various genesfound to be modulated upon Q10 treatment are summarized in Table 27below. The results showed that each of the genes ANGPTL3, ANGPTL4,CXCL1, CXCL3, CXCL5, ENG, MMP2 and TIMP3 were upregulated at 16 hoursfollowing treatment (by approximately 5.5, 3, 3, 3.2, 3, 3, 1 and 6.5fold, 6 fold and 5 fold, respectively, over that of control). ID3 wasdownregulated at 16 hours following Q10 treatment, by approximately 5fold over control. At 48 hours following treatment, ANGPTL3, CXCL1,CXCL3, ENG and TIMP3 were still upregulated (by approximately 3.5, 1.5,3.175, 2 and 3 fold, respectively, over control), while ANGPTL4, CXCL5,ID3 and MMP2 were downregulated by approximately 1, 1, 2 and 18 fold,respectively, over control.

TABLE 27 List of genes regulated in the Angiogenesis Arrays when HepG2cells were treated with 100 μM Q10 Gene Gene Name. Gene Function.ANGPTL3 angiopoietin-like 3 Predominantly expressed in live, role incell migration and adhesion, regulates fatty acid and glycerolmetabolism. ANGPTL4 angiopoietin-like 4 Regulated by PPARG, apoptosisinhibitor for vascular endothelial cells, role lipid and glucosemetabolism and insulin sensitivity. CXCL1 chemokine (C—X—C motif) Rolein cell proliferation and migration ligand 1 (melanoma growthstimulating activity, alpha) CXCL3 chemokine (C—X—C motif) Chemokineactivation, hepatic stellar cell ligand 3 activation, migration,proliferation. CXCL5 chemokine (C—X—C motif) Produced along with IL8when stimulated ligand 5 with IL1 or TNFA. Role in chemotaxis,migration, proliferation. ENG endoglin Binds to TGFBR and is involved inmigration, proliferation, attachment and invasion. ID3 inhibitor of DNAbinding 3, Regulates MMP2, Regulated by TGFB1, dominant negative helix-Vitamin D3, Retinoic acid, VEGFA, loop-helix protein involved inapoptosis, proliferation, differentiation, migration. MMP2 matrixmetallopeptidase 2 Hepatic stellate cell activation, HIF□ (gelatinase A,72 kDa signaling, binds to TIMP3, involved in gelatinase, 72 kDa type IVtumorigenesis, apoptosis, proliferation, collagenase) invasiveness,migration and chemotaxis. TIMP3 TIMP metallopeptidase Regulates MMP2,ICAM1. Regulated by inhibitor 3 TGFB, EGF, TNF, FGF and TP53. Involvedin apoptosis, cell-cell adhesion and malignancy.

Proteins known to be involved in the process of angiogenesis werecomponents in the RT-PCR array. Angiogenesis is a critical process bywhich cancer cells become malignant. Some of these proteins are alsoimplicated in diabetes.

ANGPTL3 and ANGPTL4: The literature related to ANGPTL3 connects thisprotein to the regulation of lipid metabolism. In particular, theliterature (Li, C. Curr Opin Lipidol. 2006 April; 17(2):152-6) teachesthat both angiopoietins and angiopoietin-like proteins share similardomain structures. ANGPTL3 and 4 are the only two members of thissuperfamily that inhibit lipoprotein lipase activity. However, ANGPTL3and 4 are differentially regulated at multiple levels, suggestingnon-redundant functions in vivo. ANGPTL3 and 4 are proteolyticallyprocessed into two halves and are differentially regulated by nuclearreceptors. Transgenic overexpression of ANGPTL4 as well as knockout ofANGPTL3 or 4 demonstrate that these two proteins play essential roles inlipoprotein metabolism: liver-derived ANGPTL3 inhibits lipoproteinlipase activity primarily in the fed state, while ANGPTL4 playsimportant roles in both fed and fasted states. In addition, ANGPTL4regulates the tissue-specific delivery of lipoprotein-derived fattyacids. ANGPTL4 is thus an endocrine or autocrine/paracarine inhibitor oflipoprotein lipase depending on its sites of expression.

Lipoprotein lipase is an enzyme that hydrolyzes lipids in lipoproteins,such as those found in chylomicrons and very low-density lipoproteins(VLDL), into three free fatty acids and one glycerol molecule.Lipoprotein lipase activity in a given tissue is the rate limiting stepfor the uptake of triglyceride-derived fatty acids. Imbalances in thepartitioning of fatty acids have major metabolic consequences. High-fatdiets have been shown to cause tissue-specific overexpression of LPL,which has been implicated in tissue-specific insulin resistance andconsequent development of type 2 diabetes mellitus.

The results in this Example indicate that Q10 is modulating proteinsinvolved in lipid metabolism and thus warrants further investigation ofANGPTL3/ANGPTL4 and their related pathways. For example, ANGPTL3/ANGPTL4have been implicated to play a role in the following pathways: Akt,cholesterol, fatty acid, HDL-cholesterol, HNF1A, ITGA5, ITGA5, ITGAV,ITG83, L-trilodothynonine, LIPG, LPL, Mapk, Nrth, NR1H3, PPARD, PTK2,RXRA, triacylglerol and 9-cis-retinoic acid.

Example 14 PCR Apoptosis Array on Liver Cancer (HEPG2) Cells

Apoptosis arrays were run for samples treated with 100 uM Q10 for 16 and48 hours as described above. However, the array for 48 hours was runchoosing FAM as the fluorophore instead of SYBR. Both FAM and SYBRfluoresce at the same wavelength.

The various genes found to be modulated upon Q10 treatment aresummarized in Table 28 below. The results showed that CASP9 wasupregulated at 16 hours following Q10 treatment, by approximately 61fold over control, while BAG1 and TNFRSF1A were downregulated at 16hours following treatment by approximately 6 and 4 fold, respectively,over that of control. At 48 hours following treatment, CASP9, BAG1 andTNFRSF1A were upregulated by approximately 55, 1 and 1 fold,respectively, over control.

TABLE 28 List of genes regulated in the Apoptosis Arrays when HepG2cells were treated with 100 μM Q10 Gene Gene Name Gene Function. BAG1BCL2-associated athanogene Involved with Apoptosis CASP9 caspase 9,apoptosis-related Apoptosis through release cysteine peptidase ofcytochrome c. TNFRSF1A tumor necrosis factor anti-apoptosis, binds manyreceptor superfamily, cell death factors, regulates member 1A ICAM1

Example 15 Assessing Ability of MIM or Epi-Shifter to Treat OncologicalDisorder

The ability of a selected MIM or Epi-shifter, e.g., CoQ10, to treat anoncological disorder, e.g., melanoma, is evaluated in a murine model.Melanoma tumors are induced in mice by SK-MEL28 injection into thesubcutaneous layer. The animal study consists of both a control andtreatment group each containing four mice. The mice are inoculated withtwo tumors. A topical formulation of the MIM or Epi-shifter is appliedto the tumors in the treatment group daily for a period of 30 days,after which, the tumors are excised and the mass is determined. A MIM orEpi-shifter is identified as effective in treating the tumor when thedifference in the overall mean mass of the treatment group issignificant compared to the control.

Example 16 Identification of a MIM Associated with an OncologicalDisorder

In order to evaluate a candidate molecule (e.g., environmentalinfluencer) as a potential MIM, the selected candidate MIM isexogenously added to a panel of cell lines, including both diseased(cancer) cell lines and normal control cell lines, and the changesinduced to the cellular microenvironment profile for each cell line inthe panel are assessed. Changes to cell morphology, physiology, and/orto cell composition, including for example, mRNA and protein levels, areevaluated and compared for the diseased cells as compared to normalcells.

Changes to cell morphology/physiology are evaluated by examining thesensitivity and apoptotic response of cells to the candidate MIM. Theseexperiments are carried out as described in detail in Example 3.Briefly, a panel of cell lines consisting of at least one control cellline and at least one cancer cell line are treated with variousconcentrations of the candidate MIM. The sensitivity of the cell linesto the potential MIM are evaluated by monitoring cell survival atvarious times, and over the range of applied concentrations. Theapoptotic response of the cell lines to the potential MIM are evaluatedby using, for example, Nexin reagent in combination with flow cytometrymethodologies. Nexin reagent contains a combination of two dyes, 7AADand Annexin-V-PE, and allows quantification of the population of cellsin early and late apoptosis. An additional apoptosis assay that measuressingle-stranded DNA may be used, using for example APOSTRAND™ ELISAmethodologies. The sensitivity and apoptotic response of the disease andcontrol cell lines are evaluated and compared. A molecule that displaysdifferential cytotoxicity and/or that differentially induces theapoptotic response in the diseased cells as compared to the normal cellsis identified as a MIM.

Changes in the composition of cells following treatment with thecandidate MIM are evaluated. Changes in gene expression at the mRNAlevel are analyzed using Real-Time PCR array methodology. Theseexperiments are carried out as described in detail in Examples 6 and9-13. Briefly, the candidate MIM is exogenously added to one or morecell lines including, for example a diseased cell and a normal controlcell line, and mRNA is extracted from the cells at various timesfollowing treatment. The level of mRNAs for genes involved in specificpathways are evaluated by using targeted pathway arrays, including, forexample, arrays specific for apoptosis, oxidative stress and antioxidatedefense, angiogenesis, heat shock or diabetes. The genes that arealtered in their mRNA transcription by a two-fold level or greater areidentified and evaluated. A molecule that induces changes in mRNA levelsin cells and/or that induces differential changes in the level of one ormore mRNAs in the diseased cells as compared to the normal cells isidentified as a MIM.

In complementary experiments, changes in gene expression at the proteinlevel are analyzed by using antibody microarray methodology,2-dimensional gel electrophoresis followed by protein identificuationusing mass spectrometry characterization, and by western blot analysis.These experiments are carried out as described in detail in Examples 7,4 and 8, respectively. Briefly, the candidate MIM is exogenously addedto one or more cell lines, including, for example a diseased cell and anormal control cell line, and soluble protein is extracted from thecells at various times, e.g., 6 hours or 24 hours, following treatment.Changes induced to protein levels by the candidate MIM are evaluated byusing an antibody microarray containing antibodies for over 700proteins, sampling a broad range of protein types and potential pathwaymarkers. Further complementary proteomic analysis can be carried byemploying 2-dimensional (2-D) gel electrophoresis coupled with massspectrometry methodologies. The candidate MIM is exogenously added toone or more cell lines, including, for example a diseased cell and anormal control cell line, and cell pellets are lysed and subjected to2-D gel electrophoresis. The gels are analyzed to identify changes inprotein levels in treated samples relative to control, untreatedsamples. The gels are analyzed for the identification of spot changesover the time course of treatment due to increased levels, decreasedlevels or post-translational modification. Spots exhibitingstatistically significant changes are excised and submitted for proteinidentification by trypsin digestiona dn mass spectrometrycharacterization. The characterized peptides are searched againstprotein databases with, for example, Mascot and MSRAT software analysisto identify the proteins. In addition to the foregoing 2-D gel analysisand antibody microarray experiments, potential changes to levels ofspecific proteins induced by the candidate MIM may be evaluated byWestern blot analysis. In all of the proteomic experiments, proteinswith increased or decreased levels in the various cell lines areidentified and evaluated. A molecule that induces changes in proteinlevels in cells and/or that induces differential changes in the level ofone or more proteins in the diseased cells as compared to the normalcells is identified as a MIM.

Genes found to be modulated by treatment with a candidate MIM from theforegoing experiments are subjected to cellular and biochemical pathwayanalysis and can thereby be categorized into various cellular pathways,including, for example apoptosis, cancer biology and cell growth,glycolysis and metabolism, molecular transport, and cellular signaling.

Experiments are carried out to confirm the entry of a candidate MIM intocells, to determine if the candidate MIM becomes localized within thecell, and to determine the level and form of the candidate MIM presentin the cells. These experiments are carried out, for example, asdescribed in detail in Example 5. For example, to determine the leveland the form of the candidate MIM present in the mitochondria,mitochondrial enriched preparations from cells treated with thecandidate MIM are prepared and analyzed. The level of the candidate MIMpresent in the mitochondria can thereby be confirmed to increase in atime and dose dependent manner with the addition of exogenous candidateMIM. In addition, changes in levels of proteins from mitochondriaenriched samples are analyzed by using 2-D gel electrophoresis andprotein identification by mass spectrometry characterization, asdescribed above for total cell protein samples. Candidate MIMs that arefound to enter the cell and to be present at increased levels, e.g., inthe mitochondria, are identified as a MIM. The levels of the candidateMIM in the cell, or, for example, specifically in the mitochondria, overthe time course examined can be correlated with other observed cellularchanges, as evidenced by, for example, the modulation of mRNA andprotein levels for specific proteins.

Candidate MIMs observed to induce changes in cell composition, e.g., toinduce changes in gene expression at the mRNA or protein level, areidentified as a MIM. Candidate MIMs observed to induce differentialchanges in cell morphology, physiology or cell composition (e.g.,differential changes in gene expression at the mRNA or protein level),in a disease state (e.g., cancer) as compared to a normal (e.g.,non-cancerous) state are identified as a MIM and, in particular, ashaving multidimensional character. Candidate MIMs found to be capable ofentering a cell are identified as a MIM and, in particular, as havingmultidimensional character since the candidate MIM thereby exhibits acarrier effect in addition to a therapeutic effect.

Example 17 Identification of CoQ10 as an Epi-Shifter Associated with aOncological Disorder

A panel of skin cell lines consisting of a control cell lines (primaryculture of keratinocytes and melanocytes) and several skin cancers celllines (SK-MEL-28, a non-metastatic skin melanoma; SK-MEL-2, a metastaticskin melanoma; or SCC, a squamous cell carcinoma; PaCa2, a pancreaticcancer cell line; or HEP-G2, a liver cancer cell line) were treated withvarious levels of Coenzyme Q10. The cancer cell lines exhibited analtered dose dependent response when compared to the control cell lines,with an induction of apoptosis and cell death in the cancer cells only.Detailed exemplary experiments are presented in, e.g., Example 3 herein.

Assays were employed to assess changes in the mRNA and protein levelscomposition of the above-identified cells following treatment withCoQ10. Changes in mRNA expression were analyzed using real-time PCRmicroarrays specific for each of apoptosis, oxidative stress andantioxidants, angiogenesis and diabetes. Changes in protein expressionwere analyzed using antibody microarray analysis and western blotanalysis. The results from these assays demonstrated that significantchanges in gene expression, both at the mRNA and protein levels, wereoccurring in the cell lines due to the addition of the Coenzyme Q10.Numerous genes known to be associated with or involved in cellularmetabolic processes were observed to be modulated as a result oftreatment with CoQ10. For example, expression of the nuclear receptorprotein HNF4A was found to be upmodulated in cells following Q10treatment. Expression of transaldolase 1 (TAL) was also modulated incells treated with Q10. TAL balances the levels of NADPH and reactiveoxygen intermediate, thereby regulating the mitochondrialtrans-membrande potentional, which is a critical checkpoint of ATPsynthesis and cell survival. Of particular relevance to oncologicaldisorders, numerous genes known to be associated with, e.g., apoptosis,cancer biology and cell growth, were identified as being regulated byQ10. Detailed exemplary experiments are presented in, e.g., Examples 4,6, 7, 8 and 9 herein.

Q10 is an essential cofactor for exidative phosphorylation processes inthe mitochondria for energy production. The level of Coenzyme Q10, aswell as the form of CoQ10, present in the mitochondria was determined byanalyzing mitochondrial enriched preparations from cells treated withCoQ10. The level of Coenzyme Q10 present in the mitochondria wasconfirmed to increase in a time and dose dependent manner with theaddition of exogenous Q10. The time course correlated with a widevariety of cellular changes as observed in modulation of mRNA andprotein levels for specific proteins related to metabolic and apoptoticpathways. Detailed exemplary experiments are presented in, e.g., Example5 herein.

The results described herein identified the endogenous molecule CoQ10 asan epi-shifter. In particular, the results identified CoQ10 as inducinga shift in the metabolic state, and partially restoration ofmitochondrial function, in cells. These conclusions are based on thefollowing interpretation of the data described herein and the currentknowledge in the relevant art.

Q10 is known to be synthesized, actively transported to, enriched in,and utilized in the mitochondrial inner membrane. Q10 is also known tobe an essential cofactor for oxidative phosphorylation processes in themitochondrial for energy production.

However, most cancer cells predominantly produce energy by glycolysisfollowed by lactic acid fermentation in the cytosol, rather than byoxidation of pyruvate in mitochondria like most normal cells. Theoxidative phosphorylation involves the electron transport complexes andcytochrome c. Apoptosis involves the disruption of the mitochondria,with permiabilization of the inter mitochondrial membrane bypro-apoptitic factors. By utilizing a different metabolic energysynthesis pathway, cancer cells are able to mitigate the normalapoptosis response to abnormalities in the cell. While not wishing to bebound by theory, Applicants propose that Q10 is functioning byupregulating the oxidative phosphorylation pathway proteins, thusswitching the mitochondrial function back to a state that wouldrecognize the oncogenic defects and trigger apoptosis. Thus, Q10 isacting as an Epi-shifter by shifting the metabolic state of a cell.

Example 18 Identification of an Epi-Shifter Associated with anOncological Disorder

A panel of skin cell lines consisting of control cell lines (e.g.,primary culture of keratinocytes and melanocytes) and cancer cell lines(e.g., SK-MEL-28, a non-metastatic skin melanoma; SK-MEL-2, a metastaticskin melanoma; or SCC, a squamous cell carcinoma; PaCa2, a pancreaticcancer cell line; or HEP-G2, a liver cancer cell line) are treated withvarious levels of a candidate Epi-shifter. Changes to cellmorphology/physiology are evaluated by examining the sensitivity andapoptotic response of cells to the candidate Epi-shifter. Theseexperiments are carried out as described in detail in Example 3.Briefly, the sensitivity of the cell lines to the candidate Epi-shifterare evaluated by monitoring cell survival at various times, and over arange of applied concentrations. The apoptotic response of the celllines to the candidate Epi-shifter are evaluated by using, for example,Nexin reagent in combination with flow cytometry methodologies. Nexinreagent contains a combination of two dyes, 7AAD and Annexin-V-PE, andallows quantification of the population of cells in early and lateapoptosis. An additional apoptosis assay that measures single-strandedDNA may be used, using for example Apostrand™ ELISA methodologies. Thesensitivity and apoptotic response of the disease and control cell linesare evaluated and compared. Candidate Epi-shifters are evaluated basedon their ability to inhibit cell growth preferentially or selectively incancer cells as compared to normal or control cells. CandidateEpi-shifters are further evaluated based on their ability topreferentially or selectively induce apoptosis in cancer cells ascompared to normal or control cells.

Assays are employed to assess changes in the mRNA and protein levelcomposition of the above-identified cells following treatment with thecandidate Epi-shifter. Changes in mRNA levels are analyzed usingreal-time PCR microarrays. These experiments are carried out asdescribed in detail in Examples 6 and 9-13. Briefly, mRNA is extractedfrom the cells at various times following treatment. The level of mRNAsfor genes involved in specific pathways are evaluated by using targetedpathway arrays, including, arrays specific for apoptosis, oxidativestress and antioxidate defense, angiogenesis, heat shock or diabetes.The genes that are altered in their mRNA transcription by a two-foldlevel or greater are identified and evaluated.

Changes in protein expression are analyzed using antibody microarrayanalysis, 2-D gel electrophoresis analysis coupled with massspectrometry characterization, and western blot analysis. Theseexperiments are carried out as described in detail in Examples 7, 4 and8, respectively. Briefly, soluble protein is extracted from the cells atvarious times, e.g., 6 hours or 24 hours, following treatment with thecandidate Epi-shifter. Changes induced to protein levels by thecandidate Epi-shifter are evaluated by using an antibody microarraycontaining antibodies for over 700 proteins, sampling a broad range ofprotein types and potential pathway markers. Further complementaryproteomic analysis can be carried out by employing 2-dimensional (2-D)gel electrophoresis coupled with mass spectrometry methodologies. Thecandidate Epi-shifter is exogenously added to the cell lines and cellpellets are lysed and subjected to 2-D gel electrophoresis. The gels areanalyzed to identify changes in protein levels in treated samplesrelative to control, untreated samples. The gels are analyzed for theidentification of spot changes over the time course of treatment due toincreased levels, decreased levels or post-translational modification.Spots exhibiting statistically significant changes are excised andsubmitted for protein identification by trypsin digestion and massspectrometry characterization. The characterized peptides are searchedagainst protein databases with, for example, Mascot and MSRAT softwareanalysis to identify the proteins. In addition to the foregoing 2-D gelanalysis and antibody microarray experiments, potential changes tolevels of specific proteins induced by the candidate MIM may beevaluated by Western blot analysis. In all of the proteomic experiments,proteins with increased or decreased levels in the various cell linesare identified and evaluated.

Candidate Epi-shifters are evaluated based on changes induced to geneexpression, at the mRNA and/or protein levels, in the cell lines due tothe addition of the candidate Epi-shifter. In particular, candidateEpi-shifters are evaluated based on their ability to modulate genesknown to be associated with or involved in cellular metabolic processes.Of particular relevance to oncological disorders, candidate Epi-shiftersare evaluated based on their ability to modulate genes known to beassociated with, for example, apoptosis, cancer biology and cell growth.

The level of the candidate Epi-shifter, as well as the form of thecandidate Epi-shifter, present in the cell or a particular cell locationis determined using routine methods known to the skilled artisan. Forexample, the level of the candidate Epi-shifter in mitochondria overtime and over a range of doses is determined by analyzing mitochondrialenriched preparations from cells treated with the candidate Epi-shifter.The levels of the candidate Epi-shifter in the mitochondria over thetime course can be compared and correlated with other cellular changesobserved, such as modulation of mRNA and protein levels for specificproteins related to metabolic and apoptotic pathways.

Candidate Epi-shifters observed to induce a shift in the metabolic stateof a cell based on the results obtained from the foregoing experimentsare identified as Epi-shifters. For example, a candidate Epi-shifterthat displays cytotoxicity and/or that induces apoptosis in a cell isidentified as an Epi-shifter. Preferably, a candidate Epi-shifter thatdisplays differential cytotoxicity and/or that differentially inducesthe apoptotic response in diseased (cancer) cells as compared to normalcells (e.g., Epi-shifters that differentially modulate expression ofproteins involved in apoptosis in cancer cells as compared to normalcells) is identified as an Epi-shifter.

Example 19 Identification of Vitamin D3 as an Epi-Shifter

Vitamin D3, or 1α,25-dihydroxyvitamin D3 (also known as calcitriol), isa vitamin D metabolite that is synthesized from vitamin D by a two-stepenzymatic process. Vitamin D3 interacts with its ubiquitous nuclearvitamin D receptor (VDR) to regulate the transcription of a widespectrum of genes involved in calcium and phosphate homeostasis as wellas in cell division and differentiation. Vitamin D3 has been reported tohave anticancer effects in numerous model systems, including squamouscell carcinoma, prostate adenocarcinoma, cancers of the ovary, breastand lung (reviewed in Deeb et al. 2007 Nature Reviews Cancer 7:684-700).

The anticancer effects of vitamin D3 are reported to involve multiplemechanisms, including growth arrest at the G1 phase of the cell cycle,apoptosis, tumor cell differentiation, disruption of growthfactor-mediated cell survival signals, and inhibition of angiogenesisand cell adhesion (reviewed in Deeb et al. 2007 Nature Reviews Cancer7:684-700). For example, with particular respect to apoptosis, VitaminD3 has been reported to induce apoptosis by regulating key mediators ofapoptosis, such as repressing the expression of the anti-apoptotic,pro-survival proteins BCL2 and BCL-XL, or inducing the expression ofpro-apoptotic proteins (e.g., BAX, BAK and BAD) (Deeb et al. 2007). In afurther example, with particular respect to angiogenesis, Vitamin D3 hasbeen reported to inhibit the proliferation of some tumor-derivedendothelial cells and to inhibit the expression of vascular endothelialgrowth factor (VEGF) that induces angiogenesis in tumors (reviewed inMasuda and Jones, 2006 Mol. Cancer. Ther. 5(4): 797-8070). In anotherexample, with particular respect to cell cycle arrest, Vitamin D3 hasbeen reported to induce gene transcription of the cyclin-dependentkinase inhibitor p21WAFI/CIPI and to induce the synthesis and/orstabilization of the cyclin-dependent kinase inhibiotor p27KIPI protein,both of which are critical for induction of G1 arrest. (Deeb et al.2007).

Based on the foregoing observations, Vitamin D3 is identified as anEpi-shifter, i.e., owing to its ability to shift the metabolic state ofa cell. Vitamin D3 is an Epi-shifter owing to its ability to induceapoptosis in a cell and, in particular, based on its ability todifferentially inhibit cell growth and induce the apoptotic response indiseased (cancer) cells as compared to normal cells (e.g.,differentially modulate expression of proteins, such as BCL-2, BCL-XL,and BAX, involved in apoptosis in cancer cells as compared to normalcells).

Example 20 Relative Sensitivities of Oncogenic and Normal Cells toCoenzyme Q10

The effects of Coenzyme Q10 treatment on a variety of oncogenic andnormal cell lines were examined and compared. The sensitivity of cellsto Coenzyme Q10 was assessed by monitoring induction of apoptosis. CoQ10treatment of cells was carried out as described in detail below in theMaterials and Methods. Induction of apoptosis was assessed in thetreated cells by monitoring indicators of early apoptosis (e.g., Bcl-2expression, caspase activation and by using annexin V assays) asdescribed below. From these studies, the minimal CoQ10 dosage, e.g.,concentration of CoQ10 and time of treatment, required to induceapoptosis in the panel of cell lines was determined.

In an unexpected and surprising result, the data demonstrated thatefficacy of Coenzyme Q10 treatment was greater in cell types thatexhibited increased oncogenicity and/or greater metastatic potential,i.e., cell types that were derived from more aggressive cancers ortumors. The results of these studies are summarized below in Table 29.The data demonstrates that CoQ10 is more effective in both a time andconcentration dependent manner on cells in a more aggressive cancerstate. Moreover, a surprising divergent effect was observed on normalcells as compared to oncogenic cells. Specifically, Coenzyme Q10 wasunexpectedly found to exhibit a slightly supportive role in a normaltissue environment, wherein increased proliferation and migration wasobserved in normal cells, including keratinocytes and dermalfibroblasts.

The effect of Coenzyme Q10 on gene regulatory and protein mechanisms incancer is different in a normal cell. Key cellular machinery andcomponents, such as membrance fluidity, transport mechanisms,immunomodulation, angiogenesis, cell cycle control, genomic stability,oxidative control, glycolytic flux, metabolic control and integrity ofextracellular matrix proteins, are dysregulated and thus the genetic andmolecular fingerprint of the cell is altered. The disease environmentfavors governance of cellular control processes. The data providedherein suggests that CoQ10 exerts a greater level of efficacy (e.g., incancer cells vs. normal cells, and in cells of a more aggressive cancerstate as compared to cells) of a less aggressive or non-aggressivecancer state) by normalizing some of the key aforementioned processes ina manner that allows for restored apoptotic potential.

TABLE 29 Minimal CoQ10 concentration and treatment time required forinduction of early apoptosis in various cell types Indication of EarlyLevel of apoptosis aggressiveness: (Bcl-2, 1 = normal annexin V, tissueTissue Origin or caspase Concentration Time 2 = malignant (Cell type)activation) (μM) (hr) 3 = metastatic SKIN: Keratinocytes None N/A N/A 1(Heka, Hekn) Fibroblasts None N/A N/A 1 (nFib) Melanocytes None N/A N/A1 (Hema, LP) Melanoma Strong 20 24 2 (Skmel 28) Melanoma Very Strong 2524 3 (Skmel 2) SCC, Squamous Very Strong 25 24 3 cell carcinoma BREAST:MCF-7 Strong 50 48 2 SkBr-3 Very Strong 50 24 3 BT-20 Strong 100 48 2ZR-75 Slight 200 72 2 MDA MB 468 Strong 100 48 2 Mammary None N/A 1fiboblasts: 184A1 and 184B5) (Lawrence Berkeley) PROSTATE: PC3 VeryStrong 25 24 3 LIVER: HepG2 Very Strong 50 24 3 Hep3B Very Strong 50 243 BONE: Osteosarcoma Very Strong 50 48 2 (143b) Ewing's sarcomaExtremely 5 1 3 (NCI) strong PANCREAS: 3 PaCa2 Very Strong 25 24 Heart:Aortic smooth None N/A N/A 1 muscle (HASMC)

Materials and Methods Cell Preparation and Treatment Cells Prepared inDishes or Flasks

Cells were cultured in T-75 flasks with relevant medium supplementedwith 10% Fetal Bovine Serum (FBS), 1% PSA (penicillin, streptomycin,amphotericin B) (Invitrogen and Cellgro) in a 37° C. incubator with 5%CO₂ levels until 70-80% confluence was reached. To harvest cells fortreatment, flasks were primed with 1 mL Trypsin, aspirated, trypsinizedwith an additional 3 mL, and incubated at 37° C. for 3-5 minutes. Cellswere then neutralized with an equal volμme of media and the subsequentsolution was centrifuged at 10,000 rpm for 8 minutes. The supernatantwas aspirated and the cells were resuspended with 8.5 ml of media. Amixture of 500 ul of the resuspension and 9.5 ml of isopropanol was readtwice by a coulter counter and the appropriate number of cells to beseeded into each dish was determined. Control and concentration rangingfrom 0-200 μM groups were examined in triplicate. From a 500 μM CoQ-10stock solution, serial dilutions were performed to achieve desiredexperimental concentration in appropriate dishes. Dishes were incubatedin a 37° C. incubator with 5% CO₂ levels for 0-72 hours depending oncell type and experimental protocol.

Protein Isolation and Quantification Cells Prepared in Dishes.

Following cell treatment incubation period was complete, proteinisolation was performed. Dishes of all treatment groups were washedtwice with 2 ml, and once with 1 ml of ice cold 1X Phosphate BufferedSaline (PBS). The PBS was aspirated from the dishes after the initial 2washes only. Cells were gently scraped and collected intomicrocentrifuge tubes using the final volume from the third wash andcentrifuged at 10,000 rpm for 10 minutes. After centrifugation, thesupernatant was aspirated and the pellet was lysed with 50 uL of lysisbuffer (1 uL of protease and phosphotase inhibitor for every 100 uL oflysis buffer). Samples were then frozen overnight at −20° C.

Cells Prepared in Flasks

After the cell treatment incubation period was complete, proteinisolation was performed. Flasks of all treatment groups were washedtwice with 5 mL, and once with 3 mL of ice cold 1X PBS. The PBS wasaspirated from the flasks after the first 2 washes only. Cells weregently scraped and collected into 15 mL centrifuge tubes using the finalvolume from the third wash and centrifuged for at 10,000 rpm for 10minutes. After centrifugation, the supernatant was aspirated and thepellet was lysed with an appropriate amount of lysis buffer (1 uL ofprotease and phosphotase inhibitor for every 100 uL of lysis buffer).Lysis buffer volume was dependent on pellet size. Samples weretransferred in microcentrifuge tubes and frozen overnight at −20° C.

Protein Quantification

Samples were thawed at −4° C. and sonicated to ensure homogenization theday following protein isolation. Protein quantification was performedusing the micro BCA protein assay kit (Pierce). To prepare samples forImmuno-blotting, a 1:19 solution of betamercaptoethanol (Sigma) tosample buffer (Bio-Rad) was prepared. Samples were diluted 1:1 with thebetamercaptoethanol-sample buffer solution, boiled at 95° C. for 5minutes, and frozen overnight at −20° C.

Immuno-Blotting Bcl-2, Caspase, 9, Cyotochrome c

The volume of sample to load per well was determined using the raw meanconcentration of protein obtained from the BCA protein assay.Approximately 30-60 μg of protein were loaded for each treatment timepoint. Proteins were run in triplicate on 12% Tris-HCl ready gels(Bio-Rad) or hand cast gels in 1X running buffer at 85 and 100 volts.Proteins were then transferred onto nitrocellulose paper for an hour at100 volts, and blocked for another hour in a 5% milk solution. Membraneswere placed in primary antibody (1 uL Ab:1000 uL TBST) (Cell Signaling)overnight at −4° C. The following day, membranes were washed three timesfor ten minutes each with Tris-Buffered Saline Tween-20 (TBST), andsecondary antibody (anti-rabbit; 1 uL Ab: 1000 uL TBST) was applied foran hour at −4° C. Membranes were washed again three times for tenminutes with TBST and chemoluminescence using Pico or Femto substratewas completed (Pierce). Membranes were then developed at time intervalsthat produced the best visual results. After developing, membranes werekept in TBST at'-4° C. until Actin levels could be measured.

Actin

Membranes were placed in primary Actin antibody (1 uL Ab:5000 uL TBST)(cell signaling) for 1 hour at −4° C., washed three times for tenminutes each with TBST, and secondary antibody (anti-mouse; 1 uL Ab:1000 uL TBST) was applied for an hour at −4° C. Membranes were washedagain three times for ten minutes each with TBST and chemoluminescenceusing Pico substrate was completed (Pierce). Membranes were thendeveloped at time intervals that produced the best visual results.

Annexin V assay

Cells were washed twice in PBS10X and resuspended in Binding Buffer (0.1M

HEPES, pH 7.4; 1.4 M NaCl; 25 mM CaCl₂). Samples of 100 μl were added toa culture tube with 5 μl of annexin-PE dye or 7-ADD. The cells weremixed and incubated without light at room temperature for 15 minutes.After which, 400 μl of 1X Binding Buffer was added to each sample andthey were subjected to analysis by flow cytometry.

Examples 21-25 are herein taken from International Application WO2008/116135. The contents of which are herein incorporated in itsentirety. For the sake of consistency, the table numbers in theseexamples are not renumbered, but these Table numbers do not correspondto the tables referred to in the claims.

Example 21 Method of Preparing a CoQ10 22% Concentrate which IncludesPentylene Glycol

A concentrate was produced with CoQ10 as the lipophilic bioactive agent.About 10 kilograms (kg) of polysorbate 80 was placed in a vacuum kettleand heated to a temperature of from about 50° C. to about 65° C. About8.8 kg of CoQ10 was added to the polysorbate 80 and vacuum was appliedwith the temperature maintained at from about 50° C. to about 65° C.,and the contents mixed for about 15 minutes. The resulting material maybe referred to herein as the CoQ10 phase or the first phase. The CoQ10was dissolved in the polysorbate 80 with the vacuum kettle sealed,vacuum on, and temperature of the mix of polysorbate/CoQ10 from about50° C. to about 55° C.

In a separate kettle, about 15.8 kg of water was heated to a temperatureof from about 50° C. to about 55° C. and about 0.2 kg of phenoxyethanoland about 2 kg of HYDROLITE 5® Pentylene Glycol, USP were added to thewater and mixed until clear and uniform. About 8 kg of PHOSPHOLIPON® 85Gwas then added until dispersed. The resulting material may be referredto herein as the water phase or the second phase. The water phaseachieved a uniform dispersion and hydration of the Phospholipon-typelecithin and was added to the CoQ10/Polysorbate liquid as describedbelow at a temperature from about 50° C. to about 55° C.

A Silverson in-line production scale homogenizer, similar to theSilverson L4RT model used for laboratory scale batches, was utilized tocombine the two phases described above, (i.e., the CoQ10 phase and thewater phase). Homogenization occurred using the Silverson standardemulsion head screen by mixing at full capacity (from about 7000 rpm toabout 10,000 rpm) for a total of about 5 minutes through a closedrecirculating loop and under vacuum (from about 18 mm to about 20 mm Hg)at temperatures of from about 50° C. to about 55° C. with sweepagitation until the solubilized CoQ10 was completely encapsulated anduniformly dispersed thereby creating a thick, uniform liposomaldispersion. The resulting CoQ10 concentrate possessed CoQ10 at aconcentration of about 22% by weight. The PHOSPHOLIPON® 85Gconcentration was about 8% by weight of the total composition, that is,of the combination of the two phases described above.

In separate experiments, a one kg laboratory batch of the 22% CoQ10concentrate described above was produced and samples were taken at 5minute intervals during homogenization. The particle size of theliposomes at the various sampling times was determined utilizing laserdiffraction equipment (Malvern 2000) following the manufacturer'sdirections. Details of the homogenization process and the particle sizesobtained during homogenization are set forth below in Table 1.

TABLE 1 Process Silverson Particle Approx. peak time L4RT Head Avg.particle Intensity; temp. (minutes) Speed diameter(nm) % <300 nmexposure (° C.) 5 7000 108 84.9 55 10 7000 162 57.8 65 15 7000 112 85.455 20 7000 149 67.0 62 30 7000 120 83.0 55 45 7000 107 85.0 55As can be seen from Table 1, the CoQ10 concentrate formula and processdescribed above was capable of producing liposomes with an averagediameter of 107 nm and a 15 particle distribution that included 85% ofall liposomes produced within a size of from about 59 nm to about 279nm. A short process time (about 5 minutes) produced a liposomedispersion of CoQ10 just as efficiently as a long process time (about 45minutes). As can also be seen from the above, optimal liposome particleswere obtained where the CoQ10 was not exposed to temperatures aboveabout 55° C.

Example 22 Method of Preparing a 2% Carbomer Dispersion

A cross linked acrylic acid polymer dispersion was prepared for use as aviscosity agent in a cream composition. The acrylic acid utilized,CARBOMER 940, was prepared in a 2% dispersion with the followingcomponents set forth below in Table 2:

TABLE 2 Amount Phase Trade Name CTFA Name Percent (Kg) 1 phenoxyethanolphenoxyethanol 0.500 0.0750 1 hydrolite-5 pentylene glycol 5.000 0.75002 purified water, USP water 92.500 13.8750 3 ACRITAMER 940 CARBOMER 9402.000 0.3000 Totals 100.000 15.0000

The manufacturing process was conducted as follows. The equipment wasfirst cleaned and sanitized. On a benchtop, the phase 1 ingredients weremixed until clear and uniform. The required amount of water (phase 2)was weighed and added to a phase vessel kettle of the homgenizerdescribed above in Example 1. The water was heated with a hotwater/steam jacket to a temperature of from about 60° C. to about 65° C.Phase 1 was then added to the phase 2 water with moderate agitationuntil clear and uniform. The phase 1 container was rinsed with processwater and the temperature was maintained at from about 60° C. to about65° C. The agitator was then turned on high and CARBOMER 940 powder(phase 3) was added.

The temperature was maintained at from about 60° C. to about 65° C. andmixing continued at medium-high speed of from about 500 rpm to about 800rpm until all the CARBOMER 940 powder was added. The CARBOMER powder wasadded slowly to the vortex of the mixture of phases 1 and 2. The powderwas hand sifted slowly so that the total amount of CARBOMER was added inno less than about 10 minutes.

Mixing continued at medium-high agitation until all powder wasthoroughly dispersed and no “fish-eyes” were present. The manufacturingprocess was conducted so that all of the unneutralized CARBOMER 940powder was completely dispersed to create a smooth translucentdispersion of fully hydrated CARBOMER polymer. Agitation of the batchwas high enough to create a visible vortex, but not so high to causesplashing of the batch. Adequate mixing of the batch occurred at a highspeed of from about 800 rpm to about 1300 rpm over a period of time fromabout 60 minutes to about 90 minutes. The batch temperature wasmaintained at from about 60° C. to about 65° C. at the start of mixingand from about 55° C. to about 65° C. during mixing. The elevatedtemperature assisted in dispersion of the CARBOMER polymer and helpedprevent agglomeration.

The batch was cooled to from about 25° C. to about 30° C. with chilledwater through a jacket and mixing continued with medium-high agitation.Samples were taken to determine microquality, pH, specific gravity andviscosity.

Example 23 Method of Preparing CoQ10 Creams (1.5%, 3.0% and 5.0%) Usinga CoQ10 22% Concentrate

A cream emulsion base was formed utilizing several phases forcombination with the CoQ10 concentrate possessing liposomes ofExample 1. Phases A, B, C and D were combined to form the base cream.Phase E was the CoQ10 22% concentrate of Example 1 (22% w/w CoQ10).Details of the preparation of the cream emulsion base and the subsequentaddition of the CoQ10 22% concentrate of Example 1 are set forth below.

For preparation of the cream possessing CoQ10 22% concentrate at 1.5% byweight (“CoQ10 cream 1.5%”), the procedure for combining the variousphases was as follows with the ingredients set forth below in Tables3-7:

TABLE 3 CoQ10 Cream 1.5% Amount Phase Trade Name CTFA Name Percent (g) ARITAMOLLIENT C12-15 ALKYL 5.000 1.0000 TN BENZOATE A RITA CA CETYLALCOHOL 2.500 0.5000 A RITA SA STEARYL ALCOHOL 2.000 0.4000 A RITAPRO165 GLYCERYL 4.500 0.9000 STEARATE AND PEG-100 STEARATE

Phase A (the “Oil Phase”) included C₁₂₋₁₅ alkyl benzoates, which arelight esters added for emolliency and spreadability. The cetyl alcoholand stearyl alcohol were waxes added to impart body or texture to thecream and the glyceryl stearate and PEG-100 stearate mixture was aprimary emulsifier included to form an oil-in-water (o/w) emulsion. On abenchtop, the Phase A ingredients were weighed in a vacuum kettle andheated to from about 70° C. to about 75° C. in water bath.

TABLE 4 Phase Trade Name CTFA Name Percent Amount (g) B RITA GLYCERINglycerin 2.000 0.4000 B HYDROLITE-5 pentylene glycol 2.125 0.4250 BTRANSCUTOL P ethoxydiglycol 5.000 1.0000 B phenoxyethanol phenoxyethanol0.463 0.0926 B ACRITAMER 940, water, CARBOMER 50.000 10.0000 2%dispersion 940 B purified water USP Water 11.000 2.2000

Phase B (the “Water Phase”), contained glycerine for skin moisturizationand humectancy; propylene glycol for humectancy, to aid in skinpenetration and to improve the microbiological preservation profile;ethoxydiglycol to enhance CoQ10 skin penetration of the liposomes;phenoxyethanol for microbiological preservation; purified water as thephase solvent, and CARBOMER 940 dispersion of Example 2 above to controlthe rheological properties of the cream formulas and to add stability tothe primary emulsion.

Phase B ingredients were placed in a separate mixing kettle. Theingredients were mixed with moderate sweep mixing while heating to fromabout 70° C. to about 75° C. (no vacuum). When the Phase B ingredientsreached from about 70° C. to about 75° C., Phase A ingredients wereadded at from about 70° C. to about 75° C. with moderate sweep mixing.The mixture of Phases A and B was recirculated through a Silversonhomogenizer as described above in Example 1 (standard head) andcontinued to the next part of the process.

TABLE 5 Phase Trade Name CTFA Name Percent Amount (g) C TEALAN 99%triethanolamine 1.300 0.2600 C RITALAC LA USP lactic acid 0.300 0.0600 CRITALAC NAL Sodium lactate, 2.000 0.4000 water C distilled water Water3.312 0.6624

In Phase C (the “Neutralization and Buffer Phase”), purified water actedas a solvent and a diluent for the other ingredients in this phase.Triethanolamine was the primary neutralizer of the CARBOMER acrylic acidcopolymer in the water phase (Phase B); sodium lactate solution (60% w/win water) and lactic acid were added as a buffer system to adjust andmaintain the final pH of the cream from about 5 to about 5.5, which iswithin the natural pH range of the skin.

On a benchtop, Phase C ingredients were weighed and mixed until uniformand heated to from about 60° C. to about 65° C. The Phase C mixture wasthen added to the vacuum mixing kettle containing Phases A and B withsweep mixer on medium-high.

Mixing continued while moving to the next part of the process.

TABLE 6 Phase Trade Name CTFA Name Percent Amount (g) D TITANIUMtitanium dioxide 1.000 0.2000 DIOXIDE, #3328

Phase D (the “Pigment Phase”). A water-dispersible grade of TitaniumDioxide powder was used in the formula solely for the purpose oflightening the color of the final cream color. The yellow-orange colorof the cream, imparted by CoQ10, was substantially reduced andcosmetically improved by the addition of about 1% w/w Titanium Dioxide.

For Phase D of the process, weighed TiO₂ was added to the batch (PhasesA, B and C) and mixed and recirculated through the Silverson homogenizer(high shear head) for about 10 minutes or until completely uniform andfully extended (color was checked to confirm).

It was important to ensure there was no agglomeration or clumping of thetitanium dioxide on the sweep mixing blades; this was confirmed byvisual inspection. A Silverson in line homogenizer as described above inExample 1 was used with a high shear screen to insure maximumdeagglomeration and grinding of the titanium dioxide. The finaldispersion of the titanium dioxide was checked with a Hegman PH-175fineness of grind gauge.

TABLE 7 Amount Phase Trade Name CTFA Name Percent (g) E CoQ10 WATER,7.500 1.5000 CONCENTRATE POLYSORBATE 80, 22% UBIQUINONE, (From Example 1LECITHIN, above) PENTYLENE GLYCOL, PHENOXYETHANOL Totals 100.000 20.000

Recirculation was stopped and the batch was cooled to from about 50° C.to about 55° C. with the sweep mixer on medium, at a speed of about 30rpm. The previously weighed CoQ10 22% concentrate (Phase E) from Example1 was warmed to from about 45° C. to about 50° C. and added to the batch(Phases A, B, C and D).

All phases were mixed with sweep agitation at about 60 rpm with a vacuumapplied until uniform. Temperature was maintained at about 50° C.

The batch was cooled to from about 35° C. to about 45° C. with mixing atabout 60 rpm and the application of a vacuum.

The resulting material was placed into holding containers.

For preparation of a cream possessing CoQ10 22% concentrate at 3%(“CoQ10 cream 3%”) by weight, the exact same procedure described abovefor forming the cream possessing CoQ10 22% concentrate at 1.5% (“CoQ10cream 1.5%”) by weight was followed. The materials for each phase, andthe amounts utilized, are set forth below in Tables 8-12:

TABLE 8 CoQ10 Cream 3% Amount Phase Trade Name CTFA Name Percent (g) ARITAMOLLIENT C12-15 alkyl 4.000 0.8000 TN benzoate A RITA CA cetylalcohol 2.500 0.5000 A RITA SA stearyl alcohol 2.000 0.4000 A RITAPRO165 glyceryl stearate and 4.500 0.9000 PEG-100 stearate

TABLE 9 Amount Phase Trade Name CTFA Name Percent (g) B RITA GLYCERINglycerin 2.000 0.4000 B HYDROLITE-5 pentylene glycol 2.250 0.4500 BTRANSCUTOL P ethoxydiglycol 5.000 1.0000 B phenoxyethanol phenoxyethanol0.463 0.0926 B ACRITAMER 940, water, CARBOMER 40.000 8.0000 2%dispersion 940 B purified water, water 15.000 3.0000 USP

TABLE 10 Amount Phase Trade Name CTFA Name Percent (g) C TEALAN 99%triethanolamine 1.300 0.2600 C RITALAC LA Lactic acid 0.500 0.1000 CRITALAC NAL sodium lactate, water 2.000 0.4000 C purified water 2.4870.4974 water, USP

TABLE 11 Amount Phase Trade Name CTFA Name Percent (g) D TITANIUMtitanium 1.000 0.2000 DIOXIDE, #3328 dioxide

TABLE 12 Amount Phase Trade Name CTFA Name Percent (g) E CoQ10 water,15.000 3.0000 CONCENTRATE POLYSORBATE 80, 22% ubiquinone, LECITHIN,(From Example 1 pentylene glycol, above) phenoxyethanol Totals 100.00020.000

A similar cream was prepared by using the CoQ10 22% concentrate fromExample 1 in an amount of about 25% by weight to create a cream havingCoQ10 22% concentrate at a concentration of about 5% by weight.

A summary of the contents of CoQ10 creams having 1.5% CoQ10 by weight,3% CoQ10 by weight, and 5% CoQ10 by weight are set forth below in Tables13, 14 and 15 respectively. Note that in all the formulation examplesgiven above and below for CoQ10 creams, the amount of CoQ10 22%concentrate used would actually yield a final theoretical concentrationof CoQ10 22% concentrate of about 5% above the target concentration. So,for “CoQ10 Cream 1.5%”, the actual batch amount used was 7.5% by weightof a CoQ10 22% concentrate that yielded 1.58% w/w CoQ10. The “CoQ10Cream 3%” was made with 15% by weight of the CoQ10 22% concentrate thatyielded a theoretical content of 3.15% CoQ10 by weight. The 5% excessdrug was added to extend the overall shelf life of the product andmaintain the drug content from about 90% to about 110% of the label orexpected drug content.

TABLE 13 CoQ10 CREAM, 1.5% Phase Trade Name INCI Name Percent Supplier ARITAMOLLIENT TN C12-15 alkyl benzoates 5.000 RITA A RITA CA cetylalcohol 2.000 RITA A RITA SA stearyl alcohol 1.500 RITA A RITAPRO 165glyceryl stearate 4.500 RITA and PEG-100 stearate B RITA GLYCERINEGlycerine 2.000 RITA B HYDROLITE 5 pentylene glycol 2.125 SYMRISE BTRANSCUTOL P Ethoxydiglycol 5.000 GATTEFOSSE′ B phenoxyethanolPhenoxyethanol 0.463 RITA B PURIFIED WATER deionized water 11.000 BACRITAMER 940 water, pentylene glycol, 50.000 dispersion, 2% CARBOMER940, phenoxyethanol C purified water USP water 4.212 C triethanolaminetriethanolamine 1.300 RITA C RITALAC NAL sodium lactate and water 2.000RITA C RITALAC LA USP lactic acid 0.400 RITA D TITANIUM titanium dioxide1.000 MPSI DIOXIDE #3328 E CoQ10 liposome water, POLYSORBATE 80, 7.500concentrate, 22% W/W ubiquinone, lecithin, (From Example 1) pentyleneglycol, phenoxyethanol Totals 100.000

TABLE 14 CoQ10 Cream 3% Phase Ingredient % w/w A C12-C15 Alkyl Benzoate4.000 A Cetyl Alcohol 2.000 A Stearyl Alcohol 1.500 A Glyceryl Strearate& PEG 100 Stearate 4.500 B Glycerin 2.000 B Pentylene Glycol 2.250 BEthoxydiglycol 5.000 B Phenoxyethanol 0.476 B Carbomer 40.000 B PurifiedWater 16.000 C Sodium Lactate 2.000 C Purified Water 2.474 CTriethanolamine 1.300 C Lactic Acid 0.500 D Titanium Dioxide 1.000 ECoQ10 Concentrate 22% 15.000 (From Example 1) Total: 100.000

TABLE 15 CoQ10 Cream 5% Phase Ingredient % w/w A C12-C15 Alkyl Benzoate3.000 A Cetyl Alcohol 2.000 A Stearyl Alcohol 1.500 A Glyceryl Strearate& PEG 100 Stearate 4.500 B Glycerin 2.000 B Pentylene Glycol 2.000 BEthoxydiglycol 5.000 B Phenoxyethanol 0.450 B Carbomer 35.000 B PurifiedWater 14.000 C Sodium Lactate 2.000 C Purified Water 0.750 CTriethanolamine 1.300 C Lactic Acid 0.500 D Titanium Dioxide 1.000 ECoQ10 Concentrate 22% 25.000 (From Example 1) Total: 100.000 Note: 5%manufacturing overage of CoQ10 22% concentrate was added to the CoQ10cream 1.5%, CoQ10 cream 3.0% and the CoQ10 cream 5% batches (1.5% plus0.075%, 3% plus 0.15%, and 5% plus 2.5%).

Example 24 Topical Application of a CoQ10 Cream (1.5%, 3.0% or 5.0%)

Creams possessing CoQ10 produced in Example 3 (i.e., CoQ10 cream 1.5%,CoQ10 cream 3%, and CoQ10 cream 5%) above were applied to porcine skin.The topical dose study was conducted on two pigs each, one male and onefemale. Each animal had 6 test areas; three test areas on each side. Foreach pig, one side (3 sites) was dosed once per day for 7 days, whilethe opposite test side (3 test areas) for each pig was dosed only onetime on day 1. The creams from Example 3, prepared with ethoxydiglycol,were used on the male animals. The female animals received 3 testformulas that contained the same ingredients as the samples produced inExample 3 above, except they contained 5% 1,3-butylene glycol instead of5% ethoxydiglycol. Details of these formulations made with 1,3-butyleneglycol, which possessed 1.5% CoQ10 22% concentrate by weight, 3% CoQ1022% concentrate by weight and 5% CoQ10 22% concentrate by weight, areset forth below in Tables 16, 17, and 18 respectively.

TABLE 16 CoQ10 Cream 1.5% Nominal Active Butylene Glycol Base PhaseIngredient % w/w A C12-C15 Alkyl Benzoate 5.000 A Cetyl Alcohol 2.000 AStearyl Alcohol 1.500 A Glyceryl Strearate & PEG 100 Stearate 4.500 BGlycerin 2.000 B Pentylene Glycol 2.125 B Butylene Glycol 5.000 BPhenoxyethanol 0.463 B Carbomer 50.000 B Purified Water 11.001 C SodiumLactate 2.000 C Purified Water 4.211 C Triethanolamine 1.300 C LacticAcid 0.400 D Titanium Dioxide 1.000 E CoQ10 Concentrate 22% 7.500 (FromExample 1) Total: 100.000

TABLE 17 CoQ10 Cream 3% Nominal Active Butylene Glycol Base PhaseIngredient % w/w A C12-C15 Alkyl Benzoate 4.000 A Cetyl Alcohol 2.000 AStearyl Alcohol 1.500 A Glyceryl Strearate & PEG 100 Stearate 4.500 BGlycerin 2.000 B Pentylene Glycol 2.250 B Butylene Glycol 5.000 BPhenoxyethanol 0.476 B Carbomer 40.000 B Purified Water 16.000 C SodiumLactate 2.000 C Purified Water 2.474 C Triethanolamine 1.300 C LacticAcid 0.500 D Titanium Dioxide 1.000 E CoQ10 Concentrate 22% 15.000 (FromExample 1) Total: 100.000

TABLE 18 CoQ10 Cream 5% Nominal Active Butylene Glycol Base PhaseIngredient % w/w A C12-C15 Alkyl Benzoate 3.000 A Cetyl Alcohol 2.000 AStearyl Alcohol 1.500 A Glyceryl Strearate & PEG 100 Stearate 4.500 BGlycerin 2.000 B Pentylene Glycol 2.000 B Butylene Glycol 5.000 BPhenoxyethanol 0.450 B Carbomer 35.000 B Purified Water 14.000 C SodiumLactate 2.000 C Purified Water 0.750 C Triethanolamine 1.300 C LacticAcid 0.500 D Titanium Dioxide 1.000 E CoQ10 Concentrate 22% 25.000 (FromExample 1) Total: 100.000

All animals received the same dose of each formulation, which was 200mg, to a 121 cm² application area applied once or daily for 7 days.

After application, skin samples were obtained and analyzed as follows.The skin test area was gently washed with a mild soap and water mixture(e.g., 1% Ivory Soap in water or equivalent) to remove any residualtopical test formulation. If the area to be excised was larger than thedosed area, the dosed area was demarked with indelible ink to delineatethe skin area that was dosed. A full thickness skin section was removedby scalpel with a size approximating 10 cm×10 cm, to the depth andincluding the adipose layer. Following excision, the skin section waslaid flat and wrapped in two layers of plastic wrap (SARAN WRAP™ orequivalent), and frozen to about −70° C. or colder in a timely manner.Each skin section was identified as appropriate (e.g. animalidentification, study number, date, etc.). Samples were maintained atabout −70° C. or lower until examined.

Each skin section was placed in a watertight plastic bag and thawed infrom about 30° C. to about 35° C. water baths. Once thawed, each skinsection was gently rinsed with distilled deionized water to remove anyresidual surface dose and blood. All subcutaneous tissue (e.g. adipose)was removed by scalpel to the level of the papular dermis.

Each skin section was then tape stripped (TRANSPORE™, from 3M) fromabout 10 to about 20 times until approximately 10-25% surface glisteningwas observed. This process removed the stratum corneum and any residualsurface dose.

On each full skin sheet, 6 areas were demarcated with ink. Thedemarcated areas were 1 cm² in area.

Each skin section was placed in a watertight plastic bag and immersed ina .about.65° C. (.+/−0.3° C.) water bath to initiate the separationprocess of the epidermis from the dermis. The test sites were thenexcised from the skin sheet by punch, and the epidermis removed from thedermis by forceps. The individual skin sections were weighed and theweight recorded. The individual skin sections were minced with ascalpel, placed into pre-labeled tubes, and saved for subsequentanalysis.

The skin samples were extracted in isopropanol (IPA) on a shaker forabout 47 hours, then stored at about −20° C. until further processed.The samples were then centrifuged at about 13,500 rpm for about 10minutes and the supernatant was collected into 2 mL amber vials.

Quantification of CoQ10 was performed by High Performance LiquidChromatography (HPLC-UV). Briefly, HPLC was conducted on aHewlett-Packard 1100 Series HPLC system with an Agilent 1100 SeriesLC/MSD. A solvent system including about 65% Ethanol and about 35%Methanol was run through an Aquasil C18 column (about 3 mm.times.about100 mm, 5. mu.) at a flow rate of about 1 mL/min. Ten microliters ofsample were injected. Peak areas were quantified to concentration usingan external standard curve prepared from the neat standard. The curvewas spiked into IPA due to solubility issues of CoQ10 in water.

The results for the content of CoQ10 in mini-pig skin are summarized inFIGS. 1 and 2, and Tables 19 and 20 below. The 6 replicates per skinsection were corrected to tissue weight and averaged to obtain a meanfor each dosed site.

TABLE 19 Mean ± SD Tissue Weight (n = 42) Donor # Epidermis (grams)Dermis (gm) 5061873 (Male) 0.037 ± 0.012 0.682 ± 0.129 5061521 (Female)0.026 ± 0.007 0.603 ± 0.090

TABLE 20 Mean: ±SD Measured Concentration of CoQ10 in Porcine Skin (n =6/section) Donor Dose Epidermis Dermis # Sex Side (mg) (μg/gm) (μg/gm)5061873 Male Left 1.5 137.7 ± 58.2 0.72 ± 1.12 5061873 Male Left 3.0188.7 ± 40.3 <LLQ 5061873 Male Left 5.0 163.4 ± 39.1 0.16 ± 0.39 5061873Male Right 1.5  519.3 ± 101.2 0.93 ± 0.81 5061873 Male Right 3.0  315.3± 227.0 <LLQ 5061873 Male Right 5.0  331.2 ± 128.7 <LLQ 5061873 MaleCenter 0  24.6 ± 11.5 <LLQ 5061521 Female Left 1.5 135.6 ± 39.2 <LLQ5061521 Female Left 3.0 211.8 ± 60.5 <LLQ 5061521 Female Left 5.0 211.9± 67.8 <LLQ 5061521 Female Right 1.5 118.4 ± 32.6 <LLQ 5061521 FemaleRight 3.0  84.7 ± 24.6 <LLQ 5061521 Female Right 5.0 118.1 ± 26.6 <LLQ5061521 Female Center 0  25.7 ± 21.8 <LLQ <LLQ = below lower level ofquality validation range (i.e., not detected)

The data indicated that measurable amounts of CoQ10 were observed in allepidermal samples and in selected dermal samples.

All dosed sites for the epidermis were found to contain CoQ10 at levelsthat were significantly greater than the non-dosed sites (p<0.001).

There were no significant differences between the epidermal contents forCoQ10 across the three dosing concentrations in either the male orfemale pig skin sections (p>0.02)

Between the male and female pig, for the sites from the animal's rightside (1-day dosing), the epidermal content for the 1.5% CoQ10 and 5%CoQ10 applied doses from the male's skin was significantly greater thanthat seen in the female's skin (p<0.003), but not for the 3% CoQ10 dose(p=0.0329). Thus, as can be seen from the data, the penetration of theCoQ10 on a single dose basis was significantly greater for theethoxydiglycol formula vs. the butylene glycol formula (p<0.003 for the1.5% and 5% doses and p=0.0329 for the 3% dose).

The epidermal levels for both male and female skin sections, for allthree dose applications, for the 7-day dosing period (left side), werestatistically identical.

Dermal content was only observed in the male skin sections for the 1.5%CoQ10 and 5% CoQ10 dose applications from the 7-day dosing period (leftside), and the 1.5% CoQ10 dose application from the 1-day dosing period(right side).

A summary of the data is provided as follows in Table 21:

TABLE 21 % Concentration 1.5 3 5 μg drug/mg formulation 15 30 50 Amountapplied (mg): 200 200 200 Total drug applied (μg) 3000 6000 10000 Areaapplied (cm2) 121 121 121 μg Drug/cm² 24.79 49.59 82.64 Male Left side(x7 d) Epidermis (μg/cm²) 3.470 6.688 7.311 % Dose/cm² 14.0 13.5 8.8Dermis (μg/cm²) 0.575 0 0.106 % Dose/cm² 2.3 0.0 0.1 Male Right side (x1d) Epidermis (μg/cm²) 18.309 8.215 10.986 % Dose/cm² 73.8 16.6 13.3Dermis (μg/cm²) 0.582 0 0 % Dose/cm² 2.3 0.0 0.0

If one were to extrapolate the data from Table 21 to the total area ofskin, the penetration of the CoQ10 would be as set forth below in Table22.

TABLE 22 If expanded out to total area: 1.5 3 5 Epidermis 419.87 809.248884.631 (μg/121 cm²) % Dose 14.0 13.5 8.8 Epidermis 2215.389 994.0151329.306 (μg/121 cm²) % Dose 73.8 16.6 13.3

A single application of the CoQ10 cream formulation delivered an averageof 12%, 17%, or 70% of the applied dose for the respective 5%, 3%, and1.5% CoQ10 cream formulations. In general, the penetration of the CoQ10on a single dose basis was significantly greater for the ethoxydiglycolformula vs. the butylene glycol formula (p<0.003 for the 1.5% and 5%doses and p=0.0329 for the 3% dose). The data indicated that there was arise in epidermal content with applied concentration to 3% CoQ10 withthe 5% CoQ10 dose being essential equal to the 3% CoQ10 dose. Thissuggests that the skin became saturated with CoQ10 at the 3% CoQ10 dose,or that the vehicle was unable to deliver more CoQ10 above the 3% CoQ10concentration. It can be seen that the levels achieved in the skinfollowing 7 days of topical application were identical between the 2animals.

For the ethoxydiglycol formulations, and for the single applicationdata, average penetration of 73.8%, 16.6%, and 13.3% for the respective1.5%, 3% and 5% ethoxydiglycol containing creams was obtained.

An interesting and unexpected finding was the disproportional amount ofCoQ10 found in the epidermis for the 1.5% cream, the lowest dose ofCoQ10 tested. Without wishing to be bound by any theory, this enhancedpenetration of CoQ10 may be a function of the ratio of CoQ10 toethoxydiglycol in the cream formulations, or may possibly be related tothe ratio of ethoxydiglycol to CoQ10 and the phospholipid liposome. Therelatively higher ratio of ethoxydiglycol to CoQ10 used in the creamcontaining a lower concentration of CoQ10 may be responsible for thehigher amounts of CoQ10 found in the epidermis.

The 1.5% cream and 3% cream also successfully completed 9 weeksaccelerated testing (storage at about 35° C. and about 50° C.); passed 5freeze-thaw cycles packaged in both plastic jar and metal tubepackaging; and passed USP microbiological challenge testing. Resultswere confirmed for the same system with multiple development batches andat 1.5%, 3% and 5% by weight concentrations of CoQ10 in the creamprototype formulation base.

Example 25 Method of Forming CoQ10 Creams (1.5%, 3.0% and 5.0%) Using aCoQ10 21% Concentrate

Creams were produced as described in Example 3 above, except propyleneglycol was utilized instead of pentylene glycol (1,2-pentane diol;Hydrolite-5, Symrise). A concentrate was first produced as described inExample 1 above, with the components listed below in Table 23:

TABLE 23 Batch Formula - CoQ 10 Concentrate Theoretical Quantity PhaseRaw Material Name % w/w kg A Polysorbate 80 NF 25.000 5.000 AUbidecarenone USP 21.000 4.200 B Propylene Glycol USP 10.000 2.000 BPhenoxyethanol NF 0.500 0.100 C Purified Water USP 35.500 7.100 CLecithin NF 8.000 1.600 Totals 100.000 20.000

The resulting CoQ10 concentrate (CoQ10 21% concentrate) possessed CoQ10at a concentration of about 21% by weight.

A CARBOMER dispersion was prepared as described in Example 2 above foruse in forming the cream with the components listed below in Table 24:

TABLE 24 Batch Formula - Carbomer Dispersion Theoretical Quantity PhaseRaw Material Name % w/w Kg A Phenoxyethanol NF 0.500 0.0900 A PropyleneGlycol USP 5.000 0.9000 B Purified Water USP 92.500 16.6500 C Carbomer940 NF 2.000 0.3600 Totals 100.000 18.000

A cream having 1.5% by weight CoQ10 21% concentrate and another creamhaving 3% by weight CoQ10 21% concentrate were prepared as describedabove in Example 3, with the components listed below in Tables 25 and26:

TABLE 25 Batch Formula - CoQ1O Cream 1.5% Theoretical Quantity Phase RawMaterial Name % w/w kg A AlkylC12-15BenzoateNF 5.000 1.000 A CetylAlcohol NF 2.000 0.400 A Stearyl Alcohol NF 1.500 0.300 A GlycerylStearate/PEG-100 Stearate 4.500 0.900 B Glycerin USP 2.000 0.400 BPropylene Glycol USP 1.750 0.350 B Diethylene Glycol Monoethyl Ether NF5.000 1.000 B Phenoxyethanol NF 0.463 0.093 B Carbomer Dispersion, 2%50.000 10.000 B Purified Water USP 8.377 1.675 B Purified Water USP (forrinsing) 3.000 0.600 C Trolamine NF 1.300 0.260 C Lactic Acid USP 0.4000.080 C Sodium Lactate Solution USP, 60% 2.000 0.400 C Purified WaterUSP 4.210 0.842 D Titanium Dioxide USP 1.000 0.200 E CoQ10 Concentrate,21% 7.500 1.500 Totals 100.00 20.00

TABLE 26 Batch Formula - CoQ1O Cream 3% Theoretical Quantity Phase RawMaterial Name % w/w kg A AlkylC12-15BenzoateNF 4.000 0.800 A CetylAlcohol NF 2.000 0.400 A Stearyl Alcohol NF 1.500 0.300 A GlycerylStearate/PEG-100 Stearate 4.500 0.900 B Glycerin USP 2.000 0.400 BPropylene Glycol USP 1.500 0.300 B Diethylene Glycol Monoethyl Ether5.000 1.000 B Phenoxyethanol NF 0.475 0.095 B Carbomer Dispersion, 2%40.000 8.000 B Purified Water USP 13.725 2.745 B Purified Water USP (forrinsing) 3.000 0.600 C Trolamine NF 1.300 0.260 C Lactic Acid USP 0.5000.100 C Sodium Lactate Solution USP, 60% 2.000 0.400 C Purified WaterUSP 2.500 0.500 D Titanium Dioxide USP 1.000 0.200 E CoQ10 Concentrate,21% 15.000 3.000 Totals 100.000 20.000

Example 26 Method of Forming a CoQ10 21% Concentrate which IncludesPropylene Glycol

A CoQ10 21% concentrate composition was prepared by combining phases Aand B as described below. Phase A included Ubidecarenone USP (CoQ10) at21% w/w and polysorbate 80 NF at 25% w/w. Phase B included propyleneglycol USP at 10.00% w/w, phenoxyethanol NF at 0.50% w/w, lecithin NF(PHOSPHOLIPON 85G) at 8.00% w/w and purified water USP at 35.50% w/w.All weight percentages are relative to the weight of the entire CoQ1021% concentrate composition. The percentages and further details arelisted in the following table.

TABLE 27a Phase Trade Name INCI Name Percent A RITABATE 80 POLYSORBATE80 25.000 A UBIDECARENONE UBIQUINONE 21.000 B PURIFIED WATER WATER35.500 B PROPYLENE GLYCOL PROPYLENE GLYCOL 10.000 B PHENOXYETHANOLPHENOXYETHANOL 0.500 B PHOSPHOLIPON 85G LECITHIN 8.000 Totals 100.000

The phenoxyethanol and propylene glycol were placed in a suitablecontainer and mixed until clear. The required amount of water was addedto a second container (Mix Tank 1). Mix Tank 1 was heated to between 45and 55° C. while being mixed. The phenoxyethanol/propylene glycolsolution was added to the water and mixed until it was clear anduniform. When the contents of the water phase in Mix Tank 1 were withinthe range of 45 to 55° C., Phospholipon G was added with low to moderatemixing. While avoiding any foaming, the contents of Mix Tank 1 was mixeduntil the Phospholipon 85G was uniformly dispersed. The polysorbate 89was added to a suitable container (Mix Tank 2) and heated to between 50and 60° C. The Ubidecarenone was then added to Mix Tank 2. Whilemaintaining the temperature at between 50 and 60° C. Mix Tank 2 wasmixed until all the Ubidecarenone was dissolved. After all theUbidecarenone had been dissolved, the water phase was slowly transferredto Mix Tank 2. When all materials have been combined, the contents werehomogenized until dispersion is smooth and uniform. While being carefulnot to overheat, the temperature was maintained at between 50 and 60° C.The homogenization was then stopped and the contents of Mix Tank 2 weretransferred to a suitable container for storage.

Example 27 Method of Forming a 0.5 kg Batch of CoQ10 21% Concentratewhich Includes Propylene Glycol

A 0.5 kg of CoQ10 21% concentrate composition was prepared by combiningphases A and B as described below. Phase A included Ubidecarenone USP(CoQ10) at 21% w/w and polysorbate 80 NF at 25% w/w. Phase B includedpropylene glycol USP at 10.00% w/w, phenoxyethanol NF at 0.50% w/w,lecithin NF (PHOSPHOLIPON 85G) at 8.00% w/w and purified water USP at35.50% w/w. All weight percentages are relative to the weight of theentire CoQ10 cream 21% concentrate composition. The percentages, amountsand further details are listed above in the following table.

TABLE 28a Amount Phase Trade Name INCI Name Percent (Kg) A RITABATE 80POLYSORBATE 25.000 0.1250 80 A UBIDECARENONE UBIQUINONE 21.000 0.1050 BPURIFIED WATER WATER 35.500 0.1775 B PROPYLENE PROPYLENE 10.000 0.0500GLYCOL GLYCOL B PHENOXY- PHENOXY- 0.500 0.0025 ETHANOL ETHANOL BPHOSPHOLIPON LECITHIN 8.000 0.0400 85G Totals 100.000 0.5000

All equipment was clean and sanitary. Polysorbate 80 was directlyweighed in PK-2 kettle and heat in vacuum kettle PK-2 to 50-55° C. TheUbidecarenone USP was weighed on benchtop and the weight double checkedby adding to tared PK-2 vessel (agitators off). The PK-2 was closed andsealed. The closed and sealed PK-2 was mixed with sweep mixers on lowwhile maintaining 50-55° C. temperature and vacuum on for 15 minutes.The Phase was examined to insure all powder has dissolved in polysorbatebefore moving to the next step. In PK-1, the required amount of waterwas added and heated to 50-55° C. On benchtop, phenoxyethanol andHydrolite-5 were weighed and mixed until clear and uniform and water wasadded with moderate mixing until clear and uniform. When above watermixture reached 50-55° C., lecithin was added with low-moderate mixing,avoid foaming and mixed until dispersed. Water phase was transfer fromPK-1 to a 5 gallon container. With both water phase and CoQ10 phase at50-55° C., the water phase was added to CoQ10 phase with moderate sweepmixing. Once all materials have been transferred to PK-2, the batch wasrecirculated through Silverson with standard shear head at 7000 rpm for3-5 minutes with PK-2 vessel closed and vacuum on. Temperature wasmaintained at 50-55° C. and was not allowed to overheat. Recirculationwas stopped and the batch was cooled to 30-35° C. with moderate sweepmixing. The concentrate was pumped into temporary transfer containers.

Example 28 Method of Preparing a 20 kg Batch of CoQ10 21% Concentratewhich Includes Propylene Glycol

A 20 kg batch of CoQ10 21% concentrate was prepared by combining theingredients of phases A, B and C. Phase A included polysorbate 80 NF at25.00% w/w and Ubidecarenone USP at 21.00% w/w. Phase B includedpropylene glycol USP at 10.00% w/w and phenoxyethanol NF at 0.50% w/w.Phase C included purified water USP at 35.50% w/w and lecithin NF at8.00% w/w. The percentages, amounts and further details are presented inthe following table.

TABLE 29 RM Theoretical Quantity Phases Number Raw Material Name % w/wgm A RM-002 RM-002: Polysorbate 80 25.000 5,000 NF A RM-010 RM-010:Ubidecarenone 21.000 4,200 USP B RM-021 RM-021: Propylene Glycol 10.0002,000 USP B RM-013 RM-013: Phenoxyethanol 0.500 100.0 NF C RM-011RM-011: Purified Water 35.500 7,100 USP C RM-017 RM-017: Lecithin NF8.000 1,600 Totals 100.000 20,000 For purging PK-2 (Lee Vacuum Tank)RM-019 or Nitrogen 97% NF q.s. q.s. RM-020 Nitrogen NF

In preparing the 20 kg batch of CoQ10 21% concentrate, the area wascleaned and verified clean. All equipment was cleaned and withinexpiration/calibration. 100.0 gm phenoxyethanol was weighed and placedinto a clean beaker.

To prepare Phase B, two thousand grams (2,000 gm) of propylene glycolwas weighed and placed into a clean 2-L SS beaker. Further, 2000 gm ofpurified water was weighed and placed into a clean container labeled“water for rinsing.”

To the 2-L SS beaker containing the pre-weighed 2,000 gm propyleneglycol, the pre-weighed 100 gm phenoxyethanol was added. The beaker thatcontained the phenoxyethanol was rinsed into the 2-L beaker with ⅓ ofthe water for rinsing. The contents of the 2-L beaker was mixed with aspatula until clear and uniform and was labeled as Phase B.

In the following step, 1,600 gm of lecithin was weighed and 5,100 gm ofpurified water was weighed. Appropriately labeled charts were placed inthe temperature recorders TIC-1 for the PK-1 and TIC-2 for the PK-2.5,100 gm of purified water was added to the PK-1 and the water wasmanually heated in the PK-1 to 50-55° C. The agitator was turned on anda slight vortex was maintained. The Phase B solution was then slowlyadded from the 2-L SS beaker into the PK-1. The SS beaker was thenrinsed with the approximately ⅓ of the water for rinsing. The rinsatewas slowly added to the PK-1. The temperature was manually maintained at50-55° C. The lecithin NF was then slowly added and mixed until it wasdispersed. The temperature was manually maintained at 50-55° C. and themixing continued until Phase B was ready for transfer to PK-2.

To prepare Phase A, 5,000 gm of polysorbate 80 was weighed and placedinto a clean container while 4,200 gm of Ubidecarenone USP was weighed.To compound the concentrate, the equipment included a Lee Vacuum Tank(PK-2), a Silverson Homogenizer (P-2) and a Waukesha Pump (P-1). First,it was confirmed that the bottom valve of the PK-2 was closed. Thepre-weighed 5,000 gm of polysorbate 80 was then added into the PK-2through the sight glass portal. The sight glass was replaced on the PK-2after the addition was complete.

The PK-2 agitator was then turned on and the polysorbate 80 was manuallyheated in the PK-2 to 50-55° C. When the temperature of the polysorbate80 reached that temperature range, the 4,200 gm of pre-weighedUbidecarenone USP was added through the access portal on the PK-2. Aspatula was use to remove any Ubidecarenone which was caked on theagitator blades during addition. When addition was completed, the sightglass was replaced. The temperature was manually maintained at between50-55° C. and mixed for 15 minutes. The contents of the PK-2 wasinspected through the sight glass portal to evaluate if theUbidecarenone was dissolved in the polysorbate 80. The PK-1 agitator(A-1) was then turned off.

Through the access portal of the PK-2, the contents of the PK-1 (PhaseB) were added to the PK-2. The PK-1 was then rinsed with the remaining“water for rinsing.” The PK-2 was manually heated to 50-55° C. Thecontents of the PK-2 were then recirculated through the P-1 and P-2 withthe Silverson high shear screen in P-2 at maximum rpm for 5-10 minutes.The vacuum was turned on and was maintained at maximum to preventfoaming. The temperature was manually maintained at 50-55° C.

Four samples were removed: two 30 gm samples for Micro test and two 400gm samples for physico/chemical testing. One set of sample was labeledas retain. The product was then transferred into a clean HDPE closed-topcontainer. The batch size was 20,000 gm.

Example 29 Method of Preparing a CoQ10 Cream 1.5%

A CoQ10 cream 1.5% composition was prepared by combining phases A-E asdescribed below. Phase A included alkyl C₁₂₋₁₅ benzoate NF at 5.000%w/w, cetyl alcohol NF at 2.000% w/w, glyceryl stearate/PEG-100 stearateat 4.5% w/w and stearyl alcohol NF at 1.5% w/w. The percentages andamounts are reflected in the following table.

TABLE 30 Phase Trade Name CTFA Name Percent A RITAMOLLIENT C12-15 ALKYL4.000 TN BENZOATE A RITA CA CETYL ALCOHOL 2.000 A RITA SA STEARYLALCOHOL 1.500 A RITAPRO 165 GLYCERYL 4.500 STEARATE AND PEG-100 STEARATE

Phase B included diethylene glycol monoethyl ether NF at 5.000% w/w,glycerin USP at 2.000% w/w, propylene glycol USP at 1.750% w/w,phenoxyethanol NF at 0.463% w/w, purified water USP at 11.377% w/w andcarbomer dispersion 2% at 50% w/w. The percentages and amounts arereflected in the following table.

TABLE 31 Phase Trade Name CTFA Name Percent B RITA GLYCERIN GLYCERIN2.000 B PROPYLENE GLYCOL PROPYLENE GLYCOL 1.750 B TRANSCUTOL PETHOXYDIGLYCOL 5.000 B PHENOXYETHANOL PHENOXYETHANOL 0.463 B ACRITAMER940, 2% WATER, 50.000 DISPERSION PHENOXYETHANOL, PROPYLENE GLYCOL, ANDCARBOMER 940 B PURIFIED WATER USP WATER 11.377

Phase C included lactic acid USP at 0.400% w/w, sodium lactate solutionUSP at 2.000% w/w, trolamine NF at 1.300% w/w and purified water USP at4.210% w/w. The percentages and amounts are reflected in the followingtable.

TABLE 32 Phase Trade Name CTFA Name Percent C TEAlan 99% TRIETHANOLAMINE1.300 C RITALAC LA USP LACTIC ACID 0.400 C RITALAC NAL SODIUM 2.000LACTATE, WATER C DISTILLED WATER WATER 4.210

Phase D included titatinum dioxide USP at 1.000% w/w. While Phase Eincluded CoQ10 21% concentrate, 21% at 7.500% w/w. All weightpercentages are relative to the weight of the entire 1.5% CoQ10 creamcomposition. The percentages, amounts and further details are reflectedin the following tables.

TABLE 33 Phase Trade Name CTFA Name Percent D TITANIUM TITANIUM 1.000DIOXIDE, #3328 DIOXIDE

TABLE 34 Phase Trade Name CTFA Name Percent E CoQ10 21% WATER, 7.500CONCENTRATE POLYSORBATE 80, UBIQUINONE, LECITHIN, PROPYLENE GLYCOL,PHENOXYETHANOL

In preparing the CoQ10 cream 1.5% composition, Phase A ingredients wereadded to a suitable container and heated to between 70 and 80° C. in awater bath. Phase B ingredients, excluding the Carbomer Dispersion, wereadded to a PK-2 Kettle and mixed with moderate sweep mixing whileheating to between 70 and 80° C. The Phase C ingredients were added to asuitable container and heated to between 70 and 80° C. in a water bath.The Phase E CoQ10 concentrate was placed in a suitable container andmelted between 50 and 60° C. using a water bath, while mixing asnecessary to assure uniformity. The Carbomer Dispersion was added to asuitable container (Mix Tank) and heated to between 70 and 80° C. whilemixing. While continuing to mix, Phase B ingredients were added to theheated Carbomer Dispersion in the Mix Tank while maintaining thetemperature. While continuing to mix, Phase C ingredients were added tothe contents of the Mix Tank while maintaining the temperature. The MixTank was continually mixed and homogenized. The mixer was then turnedoff but homogenization continued while adding the Phase D ingredient tothe Mix Tank. The mixer was then turned on and the ingredients was mixedand homogenized until completely uniform and fully extended (checkcolor). Homogenization was then stopped and the batch was cooled tobetween 50 and 60° C. The mixer was then turned off and the melted CoQ10concentrate was added to the Mix Tank. The mixer was then turned on andthe contents mixed/recirculated until dispersion was smooth and uniform.The contents of the Mix Tank was then cooled to between 45 and 50° C.The cooled contents were then transferred to a suitable container forstorage until packaging.

Example 30 Method of Preparing a 0.5 kg Batch of CoQ10 Cream 1.5%

A 1.5% CoQ10 cream composition was prepared by combining phases A-E asdescribed below. Phase A included alkyl C₁₂₋₁₅ benzoate NF at 5.000%w/w, cetyl alcohol NF at 2.000% w/w, glyceryl stearate/PEG-100 stearateat 4.5% w/w and stearyl alcohol NF at 1.5% w/w. The percentages, amountsand further details are reflected in the following table.

TABLE 35 Amount Phase Trade Name CTFA Name Percent (kg) A RITAMOLLIENTC12-15 ALKYL 4.000 0.0250 TN BENZOATE A RITA CA CETYL ALCOHOL 2.0000.0100 A RITA SA STEARYL ALCOHOL 1.500 0.0075 A RITAPRO 165 GLYCERYL4.500 0.0225 STEARATE AND PEG-100 STEARATE

Phase B included diethylene glycol monoethyl ether NF at 5.000% w/w,glycerin USP at 2.000% w/w, propylene glycol USP at 1.750% w/w,phenoxyethanol NF at 0.463% w/w, purified water USP at 11.377% w/w andcarbomer dispersion 2% at 50% w/w. The percentages, amounts and furtherdetails are reflected in the following table.

TABLE 36 Amount Phase Trade Name CTFA Name Percent (kg) B RITA GLYCERINGLYCERIN 2.000 0.0100 B PROPYLENE PROPYLENE 1.750 0.0088 GLYCOL GLYCOL BTRANSCUTOL P ETHOXYDIGLYCOL 5.000 0.0250 B PHENOXY- PHENOXY- 0.4630.0023 ETHANOL ETHANOL B ACRITAMER WATER, 50.000 0.2500 940, 2% PHENOXY-DISPERSION ETHANOL, PROPYLENE GLYCOL, AND CARBOMER 940 B PURIFIED WATER11.377 0.0569 WATER USP

Phase C included lactic acid USP at 0.400% w/w, sodium lactate solutionUSP at 2.000% w/w, triethanolamine NF at 1.300% w/w and purified waterUSP at 4.210% w/w. The percentages, amounts and further details arereflected in the following table.

TABLE 37 Amount Phase Trade Name CTFA Name Percent (kg) C TEAlan 99%TRIETHANOLAMINE 1.300 0.0065 C RITALAC LA USP LACTIC ACID 0.400 0.0020 CRITALAC NAL SODIUM 2.000 0.0100 LACTATE, WATER C DISTILLED WATER 4.2100.0211 WATER

Phase D included titatinum dioxide USP at 1.000% w/w. While Phase Eincluded CoQ10 concentrate, 21% at 7.500% w/w. All weight percentagesare relative to the weight of the entire 1.5% CoQ10 cream composition.The percentages, amounts and further details are reflected in thefollowing table.

TABLE 38 Amount Phase Trade Name CTFA Name Percent (kg) D TITANIUMTITANIUM 1.000 0.0050 DIOXIDE, #3328 DIOXIDE E CoQ10 21% WATER, 7.5000.0375 CONCENTRATE POLYSORBATE 80, UBIQUINONE, LECITHIN, PROPYLENEGLYCOL, PHENOXYETHANOL

In preparing the 1.5% CoQ10 cream composition, the Phase A ingredientswere weighed and heated to between 70-75° C. in a water bath. Phase Bingredients were added to a PK-2 Kettle and mixed with moderate sweepmixing while heating to between 70-75° C. When Phase B reaches 70-75° C.Phase A was added at 70-75° C. with moderate sweep mixing. Phase A and Bwere then recirculated through Silverson.

The Phase C ingredients were weighed, mixed until uniform and heated to60-65° C. Phase C ingredients were then added to the PK-2 kettle withsweep mixer on medium-high. While continuing to mix, Phase D was addedto the batch in the PK-2 kettle. The batch was continually mixed andrecirculated through the Silverson for 10 minutes or until completelyuniform and fully extended.

The circulation was discontinued and the batch was cooled to between50-55° C. with sweep mixer on medium. After warming the Phase Eingredients to between 45 and 50° C., they were added to the batch andthe batch was mixed with vacuum at moderate speed until uniform. Thetemperature was maintained at 50° C. The batch was then cooled to 30-35°C. with low-moderate mixing and vacuum. The batch was then pumped to aholding container.

Example 31 Method of Preparing a 20 kg Batch CoQ10 Cream 1.5%

A 20 kg batch of CoQ10 Cream 1.5% composition was prepared by combiningthe ingredients of Phases A-E. The weight percentages, amounts andfurther details of the ingredients for each phase are presented in thefollowing table.

TABLE 39 RM Theoretical Quantity Phase Number Raw Material Name % w/w GmA RM-026 RM-026: Capric/Caprylic 5.000 1000.0 Triglyceride A RM-003RM-003: Cetyl Alcohol NF 2.000 400.0 A RM-005 RM-005: Stearyl Alcohol NF1.500 300.0 A RM-016 RM-016: Glyceryl 4.500 900.0 Stearate/PEG-100Stearate B RM-001 RM-001: Glycerin USP 2.000 400.0 B RM-021 RM-021:Propylene Glycol 1.750 350.0 USP B RM-007 RM-007: Diethylene Glycol5.000 1000.0 Monoethyl Ether NF B RM-013 RM-013: Phenoxyethanol NF 0.46593.0 B IP-003 IP-003: Carbomer Dispersion, 50.000 10000.0 2% B RM-011RM-011: Purified Water USP 8.375 1675.0 B RM-011 RM-011: Purified WaterUSP 3.000 600.0 (for rinsing) C RM-009 RM-009: Trolamine NF 1.300 260.0C RM-006 RM-006: Lactic Acid USP 0.400 80.0 C RM-012 RM-012: SodiumLactate 2.000 400.0 Solution USP, 60% C RM-011 RM-011: Purified WaterUSP 4.210 842.0 D RM-008 RM-008: Titanium Dioxide 1.000 200.0 USP EIP-004 IP-004: CoQ10 Concentrate, 7.500 1500.0 21% Totals 100.00 20000.0For Purging PK-2 (Vacuum Tank) RM-019 or Nitrogen 97% NF or q.s. q.s.RM-020 Nitrogen NF

The chemicals were weighed with special care taken to avoid spillageonto the weighing pan. First 200 gm of titanium dioxide (Phase D) wasweighed. Then 600 gm of purified water was weighed and labeled as “waterfor rinsing—Phase B”.

In preparing Phase A, 1000 gm of Capric/Caprylic Triglyceride, 400 gm ofcetyl alcohol NF, 300 gm of stearyl alcohol NF, and 900 gm of glycerylstearate/PEG-100 were weighed. These pre-weighed Phase A ingredientswere then added to a 4-L SS beaker and the container labeled as Phase A.Note that this pre-mix must be used within 24 hours. The Phase Acontainer was then covered and put aside for later use.

In preparing Phase B, 10,000 gm of Carbomer Dispersion 2%, as in example11A, was weighed. Further, 400 gm of glycerin USP, 350 gm of propyleneglycol, 1,000 gm of diethylene glycol monoethyl ether NF and 93 gmphenoxyethanol NF were weighed. 1675 gm of purified water was weighedand the container was labeled as “purified water for Phase B”. Thesepre-weighed Phase B ingredients were added to a 10-L SS beaker andlabeled as Phase B. Note that this pre-mix must be used within 24 hours.The contents of the Phase B container were manually mixed with a spatulauntil clear and uniform. The Phase B container was then covered and putaside for later use.

In preparing Phase C, 260 gm of triethanolamine NF, 400 gm of sodiumlactate solution USP, 60%, 842 gm of purified water (labeled as“purified water for Phase C”), and 80 gm of lactic acid USP wereweighed. These pre-weighed Phase C ingredients were then added to a 2-LSS beaker in the following order: (1) 260 gm of triethanolamine, (2) 400gm sodium lactate solution, 60%, (3) 842 gm purified water USP for PhaseC and (4) 80 gm lactic acid USP. Note that this pre-mix must be usedwithin 24 hours. The contents of the Phase C container were thenmanually mixed with a spatula until clear and uniform. The Phase Ccontainer was then covered and put aside for later use.

In preparing Phase E, 1500 gm of CoQ10 21% Concentrate was weighed andcovered in a Phase E container and put aside for later use.

In preparing the 20 kg batch of CoQ10 Cream 1.5% 2 water baths arerequired. The Phase A beaker was placed into a water bath set to 70-75°C. and the contents were mixed manually with a spatula until clear anduniform. The Phase C beaker was then placed into a water bath set at60-65° C. and the contents of the Phase C beaker were mixed manuallywith a spatula until clear and uniform. Similarly, the Phase E beakerwas placed into a water bath and set to 50-55° C. The contents of thePhase E container were mixed manually with a spatula until clear anduniform.

Additional equipment used included a water bath (E-1), a Lee vacuum Tank(PK-2), a Waukesha Pump (P-1) and a square hole high shear screen forSilverson Homogenizer.

First, it was confirmed that the bottom of the valve of PK-2 was closedand that the PK-2 was properly sealed. The sight glass was then removedfrom the PK-2. The pre-weighed 10,000 gm of Carbomer Dispersion 2.0% wasthen added to the PK-2 through the sight glass portal. A spatula wasused to transfer the Carbomer Dispersion 2.0% from the walls of itscontainer. The TIC-2 (temperature recorder) for the PK-2 was then turnedon and was checked to ensure proper operation. The agitator for the PK-2(A-2) was then turned on and the Carbomer Dispersion 2.0% in the PK-2was heated with the steam jacket to 70-80° C. The vacuum was turned off.The sight glass was then removed from the PK-2 and Phase B, from the SSbeaker, was slowly added into PK-2 through the sight glass portal. ThePhase B container was then rinsed with the “water for rinsing Phase B.”The rinsate was added into the PK-2 through the sight glass portal.Phase A was then slowly added to the PK-2. A spatula was used totransfer any Phase A from the walls of the SS beaker. Note that thePhase A temperature must be between 70-80° C. when added to the PK-2.The bottom valve from the PK-2 was then opened. The P-1 (Waukesha Pump)and the P-2 (Silverson Homogenizer) were turned on and the contents ofthe PK-2 (Vacuum Tank) were homogenized. Phase C was slowly added to thePK-2 through the access port. Note that the temperature must be between70-80° C. when added to the PK-2. It was then ensured that theHomogenization was for greater than 5 minutes then the A-2 (agitator)was turned off. The pre-weighed 200.0 gm titanium dioxide of Phase D wasthen slowly sifted through a 100 mesh screen into the PK-2. A spatulawas used to dislodge any titanium dioxide which sticks to the blades ofthe PK-2.

The sight glass was then replaced and A-2 was turned on. The contentswere continued to be mixed while recirculating through P-1 (WaukeshaPump) and P-2. The contents were homogenized for 10 minutes or untilcompletely uniform and fully extended (check color). P-2 was stoppedafter 10 minutes. The contents of the PK-2 were cooled to 50-60° C. Thesight glass was removed and the melted CoQ10 21% Concentrate (Phase E,as in Example 7A) was slowly added to the PK-2. The sight glass was thenreplaced.

The contents of the PK-2 were mixed until uniform and recirculatedthrough P-1. The temperature was maintained at 50-60° C. The nitrogen NFflow was started and then the C-2 (vacuum pump) was turned on. Note thatit is best to avoid foam up of the product. The batch was then cooled to45-50° C. and both the vacuum and the nitrogen NF were turned on. Whenthe product had cooled to temperature, the C-2 was turned off and anyvacuum with the nitrogen NF was relieved. The nitrogen NF flow remainedon. The outlet valve was then purged with product before collectingsamples or packaging the product. The product was transferred intopre-weighed, clean, HDPE closed-top containers. The A-2, P-1 andnitrogen NF flow were turned off and the batch was completed andindicated on the TIC-2 chart.

Two 30 gm (minimum) samples were removed for micro testing and two 400gm (minimum) samples were removed for physico/chemical testing. One setof samples was labeled as “retain”. The filled containers were weighed.The yielded batch size was 20,000 gm.

Example 32 Method of Preparing an 18 kg Batch of Carbomer Dispersion 2%

An 18 kg batch of Carbomer Dispersion 2.0% composition was prepared bycombining phases A-C as described below. Phase A included propyleneglycol USP at 0.50% w/w and phenoxyethanol NF at 5.00% w/w. Phase Bincluded purified water USP at 92.50% w/w while Phase C includedCarbomer 940 NF at 2.00% w/w. All weight percentages are relative to theweight of the entire CoQ10 cream 2.0% composition. The percentages andamounts of the ingredients are listed in the following table.

TABLE 40 Amount Phase Trade Name CTFA Name Percent (kg) A PHENOXY-PHENOXY- 5.00 0.9 ETHANOL ETHANOL A PROPYLENE PROPYLENE 0.500 0.09GLYCOL GLYCOL B PURIFIED WATER, PURIFIED 92.500 16.65 USP WATER CACRITAMER 940 CARBOMER 2.000 0.3600 940 NF Totals 100.000 18.00

In a suitable container, the Phase A ingredients were mixed until clearand uniform. To a second container (Mix Tank) the purified water ofPhase B was added. A portion of the purified water (“water for rinse”)was retained for rinsing the Phase A container. The water in the secondcontainer was heated to between 60 and 65° C. The Phase A ingredientswere added to the water of Phase B and the “water for rinse” was used torinse the Phase A container. The contents of the Phase A container werethen mixed until clear and uniform. The mixer speed was increased whileslowly adding (sprinkle) the Carbomer 940 of Phase C to the Mix Tank.Mixing was continued until all powder had been thoroughly dispersed andno “fish-eyes” were present. The temperature was maintained between 60to 65° C. The contents were then transferred to a suitable container forstorage.

Example 33 Method of Preparing a 3 kg Batch of Carbomer Dispersion 2%

A 3 kg batch of Carbomer Dispersion 2.0% composition was prepared bycombining phases A-C as described below. Phase A included propyleneglycol USP at 5.00% w/w and phenoxyethanol NF at 0.50% w/w. Phase Bincluded purified water USP at 92.50% w/w while Phase C includedCarbomer 940 NF at 2.00% w/w. All weight percentages are relative to theweight of the entire CoQ10 cream 2.0% composition. The percentages,amounts and further details are listed in the following table.

TABLE 41 Amount Phase Trade Name CTFA Name Percent (kg) A PHENOXY-PHENOXY- 0.500 0.0150 ETHANOL ETHANOL A PROPYLENE PROPYLENE 5.000 0.1500GLYCOL GLYCOL B PURIFIED WATER, WATER 92.500 2.7750 USP C ACRITAMER 940CARBOMER 2.000 0.0600 940 Totals 100.000 3.0000

All equipment was cleaned and sanitized. On benchtop, phase Aingredients were mixed until clear and uniform. The required amount ofwater was weighed and added to Kettle PK-1 (phase vessel). The water inPK-1 was heated with hot water/steam jacket to 60-65° C. Phase A wasadded to Phase B water with moderate agitation until clear and uniform.Phase A container was rinsed with process water phase while maintainingthe temperature at 60-65° C. Mixing was continued at medium-highagitation until all powder had been thoroughly dispersed and no“fish-eyes” were present. The contents were sampled for micro quality,pH, specific gravity and viscosity. The batch was then pumped into aclean & sanitized 5 gallon closed top carboy based on pH, specificgravity and viscosity within specifications.

Example 34 Method of Preparing an 18 kg Batch of Carbomer Dispersion 2%

An 18 kg batch of Carbomer Dispersion 2% was prepared by combining theingredients of Phases A, B and C. Phase A included phenoxyethanol NF at0.5o % w/w, propylene glycol USP at 5.00% w/w, purified water USP at92.50% w/w and Carbomer 940 NF at 2.00% w/w.

TABLE 41 RM Theoretical Quantity Phase Number Raw Material Name % w/wGMS A RM-013 Phenoxyethanol NF 0.500 90.0 A RM-021 Propylene Glycol USP5.000 900 B RM-011 Purified Water USP 92.500 16,650 C RM-004 Carbomer940 NF 2.000 360 Totals 100.000 18,000

The equipment used in this batch preparation included a SartoriusBalance, a Mettler Balance, a Chart Recorder, a Lee Phase Tank and a 2-Lstainless steel (SS) beaker.

Before weighing the ingredients, the production area was cleaned andverified as being clean. All equipment were likewise cleaned andverified as clean and within expiration/calibration. The balancecalibration was checked and recorded. The weighing containers were taredto avoid spillage onto the weighing pan. First 1,650 gms of purifiedwater was weighed and placed in a container labeled “water for rinsing.”Another 15,000 gms of purified water was also weighed. 360 gms ofCarbomer 940 NF was also weighed.

Phase A was prepared by weighing 90 gms of phenoxyethanol into a beaker.900 gms of propylene glycol USP was then weighed into a 2-L SS beaker.The pre-weighed phenoxyethanol NF was then added to the 2-L SS beakercontaining the pre-weighed propylene glycol. Phenoxyethanol residueremaining in the beaker was rinsed with approximately ⅓ of the “waterfor rinsing.” The container was then labeled as Phase A. Note that thepre-mix must be used within 24 hours.

Phase A was mixed with a spatula until clear and uniform. The spatulawas removed while rinsing with ⅓ of the “water for rinsing.”

Following the preparation of Phase A, the dispersion was compoundedusing a Lee Phase Tank (PK-1). An appropriate labeled chart was placedin the TIC-1 (temperature recorder). The TIC-1 (Honeywell TemperatureRecorder) was turned on and was ensured to be operating properly. Oncethe bottom valve on the PK-1 was confirmed to be closed, the pre-weighedpurified water USP was added to the PK-1. The SS beaker was rinsed intothe PK-1 with the remaining “water for rinsing.”

The agitator was turned on to moderate and the contents of the PK-1 wereheated with the hot water/steam jacket to 60-65° C. Acceptable rangesalso includes 55-70° C. The agitator was set to the highest speedwithout causing splashing. The pre-weighed Carbomer 940 NF was evenlysifted through a 50 mesh screen into PK-1 over a period of at least 15minutes but not more than 20 minutes. The targeted temperature was60-65° C., however the acceptable range was 55-70° C. The agitator wasthen turned on to high. Mixing continued for at least 1 hour at highagitation until all powder had been thoroughly dispersed and no“fish-eyes” were present. A spatula was used to disperse any powder thatwas caught on the edge into the vortex.

Two 30 gm samples of the dispersion was removed for micro testing andtwo 400 gm sales for physico/chemical testing. One set was labeled as“retain.” The product was then transferred to clean HDPE closed-topcontainers. The resulting batch size was 18,000 gm.

Example 35 Method of Preparing a CoQ10 Cream 3% which Includes CoQ10 21%Concentrate and Alkyl Benzoate

A CoQ10 cream 3.0% composition was prepared by combining the followingphases. Phase A included alkyl C₁₂₋₁₅ benzoate NF at 4.00% w/w, cetylalcohol NF at 2.00% w/w, glyceryl stearate/PEG-100 stearate at 4.50% w/wand stearyl alcohol NF at 1.5% w/w. The percentages, amounts and furtherdetails are listed in the following table.

TABLE 43 Phase Trade Name CTFA Name Percent A RITAMOLLIENT C12-15 ALKYL4.000 TN BENZOATE A RITA CA CETYL ALCOHOL 2.000 A RITA SA STEARYLALCOHOL 1.500 A RITAPRO 165 GLYCERYL 4.500 STEARATE AND PEG-100 STEARATE

Phase B included diethylene glycol monoethyl ether NF at 5.00% w/w,glycerin USP at 2.00% w/w, propylene glycol USP at 1.50% w/w,phenoxyethanol NF at 0.475% w/w, purified water USP at 16.725% w/w andCarbomer Dispersion, 2% at 40% w/w. The percentages and amounts of theingredients are listed in the following table.

TABLE 44 Phase Trade Name CTFA Name Percent B RITA GLYCERIN GLYCERIN2.000 B PROPYLENE GLYCOL PROPYLENE GLYCOL 1.500 B TRANSCUTOL PETHOXYDIGLYCOL 5.000 B PHENOXYETHANOL PHENOXYETHANOL 0.475 B ACRITAMER940, 2% WATER, 40.000 DISPERSION PHENOXYETHANOL, PROPYLENE GLYCOL,CARBOMER 940 B PURIFIED WATER, USP WATER 16.725

Phase C included lactic acid USP at 0.50% w/w, sodium lactate solutionUSP at 2.00% w/w, Triethanolamine NF at 1.30% w/w and purified water USPat 2.50% w/w. The percentages and amounts of the ingredients are listedin the following table.

TABLE 45 Phase Trade Name CTFA Name Percent C TEALAN 99% TRIETHANOLAMINE1.300 C RITALAC LA LACTIC ACID 0.500 C RITALAC NAL SODIUM LACTATE, 2.000WATER C PURIFIED WATER 2.500 WATER, USP

Phase D included titanium dioxide USP at 1.00% w/w while Phase Eincluded CoQ10 21% concentrate, at 15.00% w/w. The percentages andamounts of the ingredients are listed in the following table.

TABLE 46 Phase Trade Name CTFA Name Percent D TITANIUM TITANIUM 1.000DIOXIDE, #3328 DIOXIDE E CoQ10 21% PROPYLENE 15 CONCENTRATE GLYCOL,POLYSORBATE 80, UBIQUINONE, WATER, PHENOXYETHANOL

All weight percentages are relative to the weight of the entire CoQ10cream 3.0% composition.

The Phase A ingredients were added to a suitable container and heated tobetween 70 and 80° C. in a water bath. The Phase B ingredients, notincluding the Carbomer Dispersion, were added to a suitable containerand mixed. The Phase C ingredients were also added to a suitablecontainer and then heated to between 70 and 80° C. in a water bath. TheCoQ10 concentrate of Phase E was placed in a suitable container andmelted between 50 and 60° C. using a water bath. The ingredients weremixed as necessary to assure uniformity. The Carbomer Dispersion wasthen added to a suitable container (Mix Tank) and heated to between 70and 80° C. while being mixed. While the ingredients were being mixed,the Phase B ingredients were added to the contents of the Mix Tank whilemaintaining the temperature. The contents were continually mixed andhomogenized. The mixer was then turned off, however, homogenization wassustained. While the homogenization continued, the titanium dioxide ofPhase D was added to the Mix Tank. The mixer was then turned on and thecontents were mixed and further homogenized until completely uniform andfully extended (check color). Homogenization was then stopped and thebatch was cooled to between 50 and 60° C. The mixer was then turned offand the melted CoQ10 concentrated was added to the Mix Tank. The mixerwas subsequently turned on and the contents mixed/recirculated untildispersion was smooth and uniform. The contents of the Mix Tank werethen cooled to between 45 and 50° C. The contents were then transferredto a suitable container for storage until unpacking.

Example 36 Method of Preparing a 0.5 kg Batch of CoQ10 Cream 3% whichIncludes CoQ10 21% Concentrate and Alkyl Benzoate

A 0.5 kg batch of CoQ10 cream 3.0% composition was prepared by combiningthe following phases. Phase A included C₁₂₋₁₅ alkyl benzoate at 4.00%w/w, cetyl alcohol NF at 2.00% w/w, glyceryl stearate/PEG-100 stearateat 4.50% w/w and stearyl alcohol NF at 1.5% w/w. The percentages andamounts are listed in the following table.

TABLE 47 Amount Phase Trade Name CTFA Name Percent (kg) A CAPRYLICC₁₂₋₁₅ alkyl benzoate 4.000 0.0200 A RITA CA CETYL ALCOHOL 2.000 0.0100A RITA SA STEARYL ALCOHOL 1.500 0.0075 A RITAPRO 165 GLYCERYL 4.5000.0225 STEARATE AND PEG-100 STEARATE

Phase B included diethylene glycol monoethyl ether NF at 5.00% w/w,glycerin USP at 2.00% w/w, propylene glycol USP at 1.50% w/w,phenoxyethanol NF at 0.475% w/w, purified water USP at 16.725% w/w andCarbomer Dispersion, 2% at 40% w/w. The percentages and amounts arelisted in the corresponding phase table below.

TABLE 48 Amount Phase Trade Name CTFA Name Percent (kg) B RITA GLYCERINGLYCERIN 2.000 0.0100 B PROPYLENE PROPYLENE 1.500 0.0075 GLYCOL GLYCOL BTRANSCUTOL P ETHOXYDIGLYCOL 5.000 0.0250 B PHENOXY- PHENOXY- 0.4750.0024 ETHANOL ETHANOL B ACRITAMER WATER, 40.000 0.2000 940, 2% PHENOXY-DISPERSION ETHANOL, PROPYLENE GLYCOL, CARBOMER 940 B PURIFIED WATER16.725 0.0836 WATER, USP

Phase C included lactic acid USP at 0.50% w/w, sodium lactate solutionUSP at 2.00% w/w, triethanolamine NF at 1.30% w/w and purified water USPat 2.50% w/w. The percentages, amounts and further details are listed inthe following table.

TABLE 49 Amount Phase Trade Name CTFA Name Percent (kg) C TEALAN 99%TRIETHANOLAMINE 1.300 0.0065 C RITALAC LA LACTIC ACID 0.500 0.0025 CRITALAC NAL SODIUM 2.000 0.0100 LACTATE, WATER C PURIFIED WATER 2.5000.0125 WATER, USP

Phase D included titanium dioxide USP at 1.00% w/w while Phase Eincluded CoQ10 21% concentrate at 15.00% w/w. The percentages, amountsand further details are listed in the following table.

TABLE 50 Amount Phases Trade Name CTFA Name Percent (kg) D TITANIUMTITANIUM 1.000 0.0050 DIOXIDE, #3328 DIOXIDE E CoQ10 21% PROPYLENE15.000 0.0750 CONCENTRATE GLYCOL, POLYSORBATE 80, WATER, UBIQUINONE,LECITHIN, PHENOXYETHANOL

All weight percentages are relative to the weight of the entire CoQ10cream 3.0% composition.

The Phase A ingredients were added to a suitable container and heated tobetween 70 and 80° C. in a water bath. The Phase B ingredients, notincluding the Carbomer Dispersion, were added to a suitable containerand mixed. The Phase C ingredients were also added to a suitablecontainer and then heated to between 70 and 80° C. in a water bath. TheCoQ10 21% concentrate of Phase E was placed in a suitable container andmelted between 50 and 60° C. using a water bath. The ingredients weremixed as necessary to assure uniformity. The Carbomer Dispersion wasthen added to a suitable container (Mix Tank) and heated to between 70and 80° C. while being mixed. While the ingredients were being mixed,the Phase B ingredients were added to the contents of the Mix Tank whilemaintaining the temperature. The contents were continually mixed andhomogenized. The mixer was then turned off, however, homogenization wassustained. While the homogenization continued, the titanium dioxide ofPhase D was added to the Mix Tank. The mixer was then turned on and thecontents were mixed and further homogenized until completely uniform andfully extended (check color). Homogenization was then stopped and thebatch was cooled to between 50 and 60° C. The mixer was then turned offand the melted CoQ10 21% concentrated was added to the Mix Tank. Themixer was subsequently turned on and the contents mixed/recirculateduntil dispersion was smooth and uniform. The contents of the Mix Tankwere then cooled to between 45 and 50° C. The contents were thentransferred to a suitable container for storage until unpacking.

Example 37 Method of Preparing a 0.5 kg Batch CoQ10 Cream 3% whichIncludes CoQ10 21% Concentrate and Caprylic/Capric Triglyceride

A 0.5 kg batch of CoQ10 cream 3.0% composition was prepared by combiningthe following phases. Phase A included Caprylic/Capric Triglyceride at4.00% w/w, cetyl alcohol NF at 2.00% w/w, glyceryl stearate/PEG-100stearate at 4.50% w/w and stearyl alcohol NF at 1.5% w/w. Thepercentages and amounts are listed in the following table.

TABLE 51 Amount Phase Trade Name CTFA Name Percent (kg) A CAPRYLICCapric Triglyceride 4.000 0.0200 A RITA CA CETYL ALCOHOL 2.000 0.0100 ARITA SA STEARYL ALCOHOL 1.500 0.0075 A RITAPRO 165 GLYCERYL 4.500 0.0225STEARATE AND PEG-100 STEARATE

Phase B included diethylene glycol monoethyl ether NF at 5.00% w/w,glycerin USP at 2.00% w/w, propylene glycol USP at 1.50% w/w,phenoxyethanol NF at 0.475% w/w, purified water USP at 16.725% w/w andCarbomer Dispersion, 2% at 40% w/w. The percentages and amounts arelisted in the corresponding phase table below.

TABLE 52 Amount Phase Trade Name CTFA Name Percent (kg) B RITA GLYCERINGLYCERIN 2.000 0.0100 B PROPYLENE PROPYLENE 1.500 0.0075 GLYCOL GLYCOL BTRANSCUTOL P ETHOXYDIGLYCOL 5.000 0.0250 B PHENOXY- PHENOXY- 0.4750.0024 ETHANOL ETHANOL B ACRITAMER WATER, 40.000 0.2000 940, 2% PHENOXY-DISPERSION ETHANOL, PROPYLENE GLYCOL, CARBOMER 940 B PURIFIED WATER16.725 0.0836 WATER, USP

Phase C included lactic acid USP at 0.50% w/w, sodium lactate solutionUSP at 2.00% w/w, triethanolamine NF at 1.30% w/w and purified water USPat 2.50% w/w. The percentages, amounts and further details are listed inthe following table.

TABLE 53 Amount Phase Trade Name CTFA Name Percent (kg) C TEALAN 99%TRIETHANOLAMINE 1.300 0.0065 C RITALAC LA LACTIC ACID 0.500 0.0025 CRITALAC NAL SODIUM 2.000 0.0100 LACTATE, WATER C PURIFIED WATER 2.5000.0125 WATER, USP

Phase D included titanium dioxide USP at 1.00% w/w while Phase Eincluded CoQ10 21% concentrate at 15.00% w/w. The percentages, amountsand further details are listed in the following tables.

TABLE 54 Amount Phases Trade Name CTFA Name Percent (kg) D TITANIUMTITANIUM 1.000 0.0050 DIOXIDE, #3328 DIOXIDE E CoQ10 21% PROPYLENE15.000 0.0750 CONCENTRATE GLYCOL, POLYSORBATE 80, WATER, UBIQUINONE,LECITHIN, PHENOXYETHANOL

All weight percentages are relative to the weight of the entire CoQ10cream 3.0% composition.

The Phase A ingredients were added to a suitable container and heated tobetween 70 and 80° C. in a water bath. The Phase B ingredients, notincluding the Carbomer Dispersion, were added to a suitable containerand mixed. The Phase C ingredients were also added to a suitablecontainer and then heated to between 70 and 80° C. in a water bath. TheCoQ10 21% concentrate of Phase E was placed in a suitable container andmelted between 50 and 60° C. using a water bath. The ingredients weremixed as necessary to assure uniformity. The Carbomer Dispersion wasthen added to a suitable container (Mix Tank) and heated to between 70and 80° C. while being mixed. While the ingredients were being mixed,the Phase B ingredients were added to the contents of the Mix Tank whilemaintaining the temperature. The contents were continually mixed andhomogenized. The mixer was then turned off, however, homogenization wassustained. While the homogenization continued, the titanium dioxide ofPhase D was added to the Mix Tank. The mixer was then turned on and thecontents were mixed and further homogenized until completely uniform andfully extended (check color). Homogenization was then stopped and thebatch was cooled to between 50 and 60° C. The mixer was then turned offand the melted CoQ10 21% concentrated was added to the Mix Tank. Themixer was subsequently turned on and the contents mixed/recirculateduntil dispersion was smooth and uniform. The contents of the Mix Tankwere then cooled to between 45 and 50° C. The contents were thentransferred to a suitable container for storage until unpacking.

Example 38 Method of Preparing a 20 kg Batch CoQ10 Cream 3% whichIncludes CoQ10 21% Concentrate and Caprylic/Capric Triglyceride

A 20 kg batch of CoQ10 Cream 3.0% composition was prepared by combiningthe ingredients of Phases A-E. The weight percentages, amounts andfurther details of the ingredients for each phase are presented in thefollowing table.

TABLE 55 RM Theoretical Quantity Phase Number Raw Material Name % w/w GmA RM-026 RM-026: Capric/Caprylic 4.000 800.0 Triglyceride A RM-003RM-003: Cetyl Alcohol NF 2.000 400.0 A RM-005 RM-005: Stearyl Alcohol NF1.500 300.0 A RM-016 RM-016: Glyceryl 4.500 900.0 Stearate/PEG-100Stearate B RM-001 RM-001: Glycerin USP 2.000 400.0 B RM-021 RM-021:Propylene Glycol 1.500 300.0 USP B RM-007 RM-007: Diethylene Glycol5.000 1000.0 Monoethyl Ether NF B RM-013 RM-013: Phenoxyethanol NF 0.47595.0 B IP-003 IP-003: Carbomer Dispersion, 40.000 8000.0 2% B RM-011RM-011: Purified Water USP 13.725 2745.0 B RM-011 RM-011: Purified WaterUSP 3.000 600.0 (for rinsing) C RM-009 RM-009: Trolamine NF 1.300 260.0C RM-006 RM-006: Lactic Acid USP 0.500 100.0 C RM-012 RM-012: SodiumLactate 2.000 400.0 Solution USP, 60% C RM-011 RM-011: Purified WaterUSP 2.500 500.0 D RM-008 RM-008: Titanium Dioxide 1.000 200.0 USP EIP-004 IP-004: CoQ10 Concentrate, 15.000 3000.0 21% Totals 100.0020000.0 For Purging PK-2 (Vacuum Tank) RM-019 or Nitrogen 97% NF or q.s.q.s. RM-020 Nitrogen NF

In preparing the 20 kg batch of CoQ10 Cream 3%, the following procedureswere followed. Before the chemical ingredients were weighed, specialcare was taken to make sure that the area and all equipment was clean.First the chemical ingredients were weighed and special care was takento avoid any spillage onto the weighing pan.

First 200 gm of titanium dioxide USP (Phase D) was weighed. Then 600 gmof purified water (labeled “water for rinsing-Phase B”) was weighed.

In preparing Phase A, 800 gm of Capric/Caprylic Triglyceride, 400 gm ofcetyl alcohol NF, 300 gm of stearyl alcohol NF, 900 gm of glycerylstearate/PEG-100 stearate were weighed. These pre-weighed Phase Aingredients were added to a 4-L SS beaker and labeled as Phase A. Notethat this premix must be used within 24 hours. The Phase A container isthen covered and put aside for later use.

In preparing Phase B, 8,000 gm of Carbomer Dispersion 2.0%, 400 gm ofglycerin USP, 300 gm of propylene glycol, 1,000 gm diethylene glycolmonoethyl Ether NF, 95 gm of phenoxyethanol NF, and 2,745 gm purifiedwater USP (labeled “Purified Water for Phase B”) were weighed. Thesepre-weighed Phase B ingredients were then added to a 10-L SS beaker.Note that this pre-mix must be used within 24 hours. The contents of thePhase B container were manually mixed using a spatula until clear anduniform. The Phase B container was then covered and put aside for lateruse.

In preparing Phase C, 260 gm of triethanolamine NF, 400 gm of sodiumlactate solution USP, 60%, 500 gm of purified water (labeled “PurifiedWater for Phase C”), and 100 gm of lactic acid USP were weighed. ThesePhase C ingredients were then added to a 2-L SS beaker in the followingorder: (1) 260 gm of trolamine, (2) 400 gm of sodium lactate solution,60%, (3) 500 gm of purified water for Phase C, and (4) 100 gm of lacticacid USP. The container was labeled Phase C. Note that this premix mustbe used within 24 hours. The contents of the Phase C container were thenmanually mixed with a spatula until clear and uniform. The Phase Ccontainer was then covered and put aside for later use.

In preparing Phase E, 3,000 gm of CoQ10 21% Concentrate was weighed andplaced in a container labeled Phase E. The Phase E container was coveredand put aside for later use.

For compounding the CoQ10 Cream 3%, 2 water baths were used to heatPhases A, C and E. First, the Phase A beaker was placed into a waterbath set to 70-75° C. and the contents were mixed manually with aspatula until clear and uniform. The Phase C beaker was placed into awater bath that was set to 60-65° C. The contents of the Phase C beakerwere manually mixed with a spatula until clear and uniform. The Phase Ebeaker was placed into a water bath and was set to a temperature of50-55° C. The contents of the Phase E beaker were manually mixed with aspatula until clear and uniform.

For compounding the cream, a water bath (E-1), a Lee Vacuum Tank (PK-2),a Waukesha Pump (P-1) and a square hole high shear screen for SilversonHomogenizer were used.

First, it was confirmed that the bottom valve of PK-2 was closed andthat the PK-2 was properly sealed. The sight glass was then removed fromthe PK-2 and the pre-weighed 10,000 gm Carbomer Dispersion 2.0% wasadded to the PK-2 tank through the sight glass portal. A spatula wasused to transfer any Carbomer Dispersion 2.0% from the walls of itscontainer. The TIC-2 (temperature recorder) for the PK-2 was then turnedon and it was ensured that the recorder was properly operational.

The agitator (A-2) for the PK-2 was turned on and the CarbomerDispersion 2.0% was heated with the steam jacket to 70-80° C. The sightglass was then removed from the PK-2 and Phase B was slowly added, fromthe SS beaker to the PK-2, through the sight glass portal. The Phase Bcontainer was then rinsed with the “water for rinsing-Phase B.” Thisrinsate was then added to the PK-2 through the sight glass portal.

Phase A was then slowly added to the PK-2. A spatula was used totransfer any Phase A remaining on the walls of the SS beaker. Note thatthe temperature of the Phase A must be between 70-80° C. when added tothe PK-2.

The bottom valve of the PK-2 was then opened and the P-1 pump and P-2(Silverson homogenizer) were turned on. The contents of the PK-2 werehomogenized.

Phase C was then slowly added to the PK-2 through the access port. Notethat the temperature must be between 70-80° C. when added. It wasensured the homogenization endured for at least 5 minutes, then the A-2agitator was turned off. The pre-weighed 200 gm titanium dioxide USP wasthen slowly sifted through a 100 mesh screen to the PK-2. A spatula wasused to dislodge any titanium dioxide that had been stuck to the bladesof the PK-2.

The sight glass was then replaced and the A-2 agitator turned on. Thecontents were continued to be mixed while recirculating through P-1 andP-2. Homogenization was allowed to continue for 10 minutes or untilcompletely uniform and fully extended. After 10 minutes P-2 was stopped.The contents of PK-2 were then cooled to 50-60° C. The sight glass wasthen removed, and the melted CoQ10 21% concentrate of Phase E was slowlyadded through the access port. The sight glass was then replaced.

The contents of the PK-2 were then mixed with A-2 until uniform. Thetemperature was maintained at 50-60° C. and the contents wererecirculated through the P-1. The nitrogen NF flow was then started andthe C-2 vacuum pump turned on. Note that avoidance of foam up of theproduct is preferred. The batch was then cooled to 45-50° C. then boththe vacuum and the nitrogen were turned on. When the product was cooledto temperature, C-2 (vacuum pump) was turned off and any vacuum wasrelieved with the nitrogen NF. The nitrogen NF flow remained on. Theoutlet valve was purged with product before collecting samples orpackaging the product.

The product was then transferred into pre-weighed, clean, HDPEclosed-top containers. The A-2 agitator, P-1 Waukesha pump and thenitrogen NF flow were turned off. The batch was completed and indicatedon the TIC-2 chart.

Two 30 gm (minimum) samples were removed for micro testing and two 400gm (minimum) samples for physico/chemical testing. One set of sampleswas labeled as “retain”. The filled containers were weighed. A batchsize of 20 kg was obtained.

Example 39 Method of Treating SCCIS by Topical Application of CoQ10Cream 3%

A CoQ10 cream 3.0% composition, as described above (e.g., examples 15and 16), was topically applied to in situ cutaneous squamous cellcarcinomas (SCCIS). Thirty five (35) subjects were topically treatedwith a 3.0% CoQ10 water-in-oil emulsion cream base medication. Themedication was shipped and stored at room temperature in light-resistantcontainers.

The analysis populations were defined as (1) Intent-to-Treat (ITT)Population, (2) Safety Population and (3) Per Protocol (PP) population.The ITT population included all subjects who were dispensed theinvestigational drug (CoQ10 3%). The Safety population included allsubjects who took at least one dose of the investigational product. ThePP population included all subjects who had SCCIS confirmed viahistological results at baseline, had a Week 6 histological examinationand did not miss any interim visits.

The subjects were otherwise healthy male or female adults with at leastone histologically confirmed non-facial SCCIS lesion. The SCCIS lesionswhich were suitable for excision, had a minimum area of 0.5 cm² and amaximum diameter of 2.0 cm, and were in a location that could beprotected from sunlight by clothing during the study. At approximatelythe same time each morning, the subject washed the lesion site thendispensed a small pea sized (50-100 mg) amount of the topical creammedication onto a piece of wax paper or applicator. The subject thenapplied the appropriate amount of cream to the lesion and surroundingarea using a cotton swab or applicator stick. The treated area was notwashed for at least 8 hours following application. At approximately thesame time each evening, the procedure was repeated. The lesion andsurrounding area was protected from sunlight with clothing.

On the first day of treatment the lesion's diameter was measured and thearea was calculated. The lesions were also photographed. At Weeks 1, 2,3, 4 and 5 the subjects were evaluated and a record was taken of theirvital signs: blood pressure, pulse rate, respiratory rate, oraltemperature. The following clinical signs/symptoms of cutaneousirritation based on the following tables were also graded as a measureof the safety of the CoQ10 3% treatment: erythema, peeling, dryness,itching, burning/stinging.

Erythema

TABLE 56 0 No observable erythema 1 Slight pinkness, limited to a smallarea 2 Mild redness over much of the treated area 3 Marked redness overmuch of the treated area 4 Severe redness, presence of edema, possibleerosion

Peeling/Scaling

TABLE 57 0 No observable scaling or peeling 1 Slight flaking oroccasional small lifting scales may be present in isolated areas 2Moderate flaking/scaling. Cracks easily evident. Edges of scales liftingover large portion of the treated area 3 Marked scaling, slightfissuring, cracking and lifting scales on most of the treated area 4Large peeling sheets of epidermis present

Dryness

TABLE 58 0 Oily shine over much of the treated area 1 Normal, nodryness, no appreciable shine 2 Slightly dry, dull appearance over asmall portion of the treated area 3 Moderately dry, very dull appearanceover much of the treated area 4 Severely dry, cracking over entiretreated area

Itching

TABLE 59 0 No itching 1 Mild itching on occasion, no impact on dailyactivities 2 Mild itching present most of the time, no impact on dailyactivities or sleep 3 Moderate itching, occasionally interferes withdaily activities or sleep 4 Intense itching that interferes with dailyactivities or sleep

Burning/Stinging

TABLE 60 0 No burning/stinging 1 Mild burning/stinging on occasion, noimpact on daily activities 2 Mild burning/stinging present most of thetime, no impact on daily activities or sleep 3 Moderateburning/stinging, occasionally interferes with daily activities or sleep4 Intense burning/stinging that interferes with daily activities orsleep

Safety evaluation by Erythema: At Baseline, 32 subjects (91.4%) had somedegree of erythema. This sign was considered slight to mild in mostsubjects (28 subjects, 80%) and 4 subjects (11.4%) had marked (Grade 3)redness. At Week 6, erythema was absent in 4 subjects (11.8%) and slightor mild in 30 subjects (88.2%), while no Grade 3 erythema was observed.The maximum erythema score observed during the study improved comparedwith Baseline in 7 subjects, did not change in 15 subjects, and worsenedin 13 subjects. The final erythema score was improved relative toBaseline in 11 subjects, was unchanged in 22 subjects, and worsened in 2subjects.

Safety evaluation by Peeling/Scaling: At Baseline, 27 subjects (77.1%)had some degree of peeling or scaling, and 8 subjects (22.9%) had none.At Week 6, visible peeling or scaling was absent for 16 subjects(47.1%), was slight in 17 subjects (50%), and was moderate in only 1subject (2.9%). The maximum score for peeling/scaling observed duringthe study improved compared with Baseline in 7 subjects, did not changein 20 subjects, and worsened in 8 subjects. The final peeling/scalingscore was improved relative to Baseline in 16 subjects, was unchanged in14 subjects, and worsened in 5 subjects.

Safety evaluation by Dryness: Eight subjects (22.9%) had slight ormoderate dryness at Baseline, while 77.1% had no dryness (Grade 1) or anoily shine (Grade 0) at the treatment area. At Week 6, all but 1 subjecthad Grade 0 or Grade 1 dryness (97.1%). The maximum dryness scoreobserved during the study improved compared with Baseline in 7 subjects,did not change in 23 subjects, and worsened in 5 subjects. The finaldryness score was improved relative to Baseline in 13 subjects, wasunchanged in 21 subjects, and worsened in 1 subject.

Safety evaluation by itching: A majority of subjects at Baselinereported no itching (74.3%) or mild, occasional itching (20%), while 2subjects (5.7%) reported itching of Grade 2 or 3. At Week 6, itching hadimproved so that 94.1% had no itching, and only 2 subjects (5.9%) hadmild, occasional itching. From Week 1 through Week 6, no subject haditching worse than Grade 1 (mild, occasional itching). The maximumitching score observed during the study improved compared with Baselinein 5 subjects, did not change in 24 subjects, and worsened in 6subjects. The final itching score was improved relative to Baseline in 9subjects, was unchanged in 24 subjects, and worsened in 2 subjects.

Safety evaluation by burning/stinging: At Baseline, burning or stingingwas absent in most subjects (94.3%) and mild in 2 subjects (5.7%). Thesescores remained virtually unchanged during the study. No subject had ascore above Grade 1 (mild), and no more than 2 subjects had a Grade 1score at any visit from Day 1 (Baseline) through Week 6. The maximumscore for burning/stinging observed during the study improved comparedwith Baseline in 2 subjects, did not change from “no burning/stinging”in 28 subjects, and worsened (from none to mild) in 5 subjects. Thefinal burning/stinging score was improved relative to Baseline in 2subjects, was unchanged in 31 subjects, and worsened in 2 subjects.

The lesions' diameters were also measured and the areas calculated toevaluate the efficacy of the CoQ10 3% treatment. At the end of the 6week treatment period there was a discontinuation visit where a recordwas taken of the vital signs and the clinical signs/symptoms weregraded. A physical examination was also performed at the 6 weekdiscontinuation visit. At the discontinuation visit fasting bloodsamples were also collected within 3 hours of the final creamapplication to determine CoQ10 plasma concentrations. The lesions werephotographed, the diameters were measured and the area was calculated.

Results of the topical treatment of SCCIS with CoQ10 cream 3% showedefficacy as depicted in the before and after photographs of FIGS. 4-9.The primary efficacy endpoint was the percentage of subjects with acomplete response defined as a negative histology assessment of thetarget lesion at Week 6. Secondary efficacy endpoints are reflected inthe percentage of subjects with a partial response, defined as at leasta 50% decrease in the area (the product of the two principal diameters)of the treated lesion at Week 6. The results showed that 23.5% of theITT population had a complete response at Week 6 while 18.5% of the PPPopulation had a complete response at Week 6.

Secondary efficacy results showed that the ITT Population had a 26.5%partial 50% response and 2.9% had a 75% partial response at Week 6. Apartial response was observed as early as Week 1 in 2 subjects and Week2 in 8 subjects. The highest partial response rates occurred at Weeks 4and 5. Interestingly, of the 8 subjects with a complete response at Week6, 4 subjects did not have a partial response based on visualinspection, and none had a 75% response. In the PP Population, 22.2% hada 50% partial response while 0% had a 75% partial response. The meanchange and mean percentage change in lesion area were −0.3 cm² and−26.1% respectively, in the ITT Population at Week 6 and −0.3 cm² and−23.4% respectively in the PP Population.

Overall, CoQ10 3% cream was safe and well tolerated. Complete cure wasachieved by approximately 25% of subjects in the ITT Population.

Example 40 Method of Treating BCC by Topical Application of CoQ10 Cream3%

Basal cell carcinoma (BCC) is the most common form of cutaneousmalignancy, and overall the most common form of cancer in the UnitedStates. The American Cancer Society estimates that over 800,000 newcases of basal cell carcinoma are diagnosed each year. Superficial basalcell carcinoma (sBCC) rarely metastasizes and is usually curable throughsurgical excision or topical agents.

A CoQ10 cream 3.0% composition, as described above in examples 35-36,was topically applied to one hundred and sixty (160) otherwise healthymale or female adults with one or more histologically confirmedsuperficial basal cell carcinoma (sBCC) lesions. One target lesion, witha minimum area of 0.5 cm² and a maximum diameter of 2.0 cm wasdesignated for treatment. The sBCC lesion was non-facial and was capableof being protected from sunlight during the study.

This study was a randomized, double-blind vehicle-controlled, parallelstudy. Each subject was randomized to one of four (4) study arms: 1.5%CoQ10 cream qd (once daily) plus vehicle cream qd (once daily), 3.0%CoQ10 cream qd (once daily) plus vehicle cream qd (once daily), 3.0%CoQ10 cream bid (twice daily), or vehicle cream bid (twice daily). Eacharm had 40 patients.

TABLE 61 AM PM 1. 3% CoQ10 3% CoQ10 2. Vehicle B 3% CoQ10 3. Vehicle A*1.5% CoQ10 4. Vehicle A Vehicle A

At an initial screening visit, the lesion diameters were measured andthe area calculated. The area is determined by measuring the two largestperpendicular diameters and multiplying for a result in cm². Thesubjects' vital signs were taken and recorded and a physical examinationwas performed. Fasting blood samples were collected and a complete bloodcount (CBC) test was performed as well as a clinical chemistry withlipid panel test. Blood samples were collected for determination ofbaseline CoQ10 plasma concentrations, duplicate samples were collectedand packaged. Urinalysis was also performed and for women ofchildbearing potential a urine pregnancy test was performed. Thesubjects were then graded for the following clinical signs/symptoms ofcutaneous irritation: erythema, peeling, dryness, itching andburning/stinging.

On the first day of the study (Day 1), the subjects' vital signs wereagain recorded and the clinical signs/symptoms of cutaneous irritationwere graded as in the screening visit. The subject was also interviewedfor adverse events, use of concurrent topical products and use ofconcurrent medications. The target sBCC lesions were then photographedand measured for diameter and area as in the screening visit. Thesubjects were then given a medication kit containing the CoQ10medication. The CoQ10 cream was applied by the patient twice daily forsix weeks to the sBCC lesion and 1 cm of surrounding skin.

The dosing regimen consisted of washing the lesion site at approximatelythe same time each morning, dispensing a small pea sized (50-100 mg)amount of topical cream from the AM tub onto a piece of wax paper orapplicator. The subject then applied the appropriate amount of cream tothe lesion and surrounding area using a Q-tip or applicator stick. Thetreated area was not washed for at least 8 hours. At approximately thesame time each evening, the procedure was repeated using topical creamfrom the PM tube.

There were interim visits by the subject which occurred at weeks 1, 2,3, 4, and 5. At each of these visits, the vital signs were recorded asat the initial treatment visit and the clinical signs/symptoms weregraded. The lesion diameter was measured and the area was calculated.The subject was then interviewed for adverse events, use of concurrenttopical products, and use of concurrent medications. Clinicalevaluations were done weekly.

At the end of the treatment period, 6 weeks, the vital signs wererecorded as at the initial treatment visit and the clinicalsigns/symptoms were graded. The lesion diameter was measured and thearea was calculated. A physical examination was also performed. Fastingblood samples were also collected after the final cream application.Blood samples were collected no more than 3 hours after the final creamapplication for determination of CoQ10 plasma concentrations. A completeblood count (CBC) test was performed and a clinical chemistry with lipidpanel test was performed.

At four weeks post-treatment (study week 10), the subject returned forfinal evaluation and excision of the sBCC lesion site. Vital signs weretaken and recorded and clinical signs/symptoms, similar to those in theinitial screening visit, were graded.

Treatment results showed, after reviewing the pathology of 110 subjects,that at least 20% of the patients who were topically treated with theCoQ10 cream 3% demonstrated diminishment of symptoms as measured by anart recognized endpoint. In particular, 24 out the 110 subjects had noevidence of sBCC based on biopsy of the lesion site at 8 weeks.

Example 41 Pharmacokinetic Results of CoQ10 Topical Treatment

Seventy-two BALB/c mice were randomly divided into nine groups of eightmice each (Groups I-IX). Group I was untreated. On day 0, groups II-VIIIwere topically treated with 0.1 g of the test article (an oil-in-watercream emulsion containing 3% w/w CoQ10 cream spiked with C-14radiolabeled API 31510) at a rate of 5 mg/cm². The radioactive API 31510was added to the 3% cream batch to yield an experimental creamformulation with a specific activity of approximately 50 μCi/g ofproduct or 5 μCi/application dose. The test article was topicallyapplied to the skin of the back of each mouse in Groups II-IX with aglass rod. Immediately following dosing the group II animals weresacrificed and a measured amount of blood, urine, feces and the targetorgans (liver, pancreas and spleen) were collected and weighed. Theblood was processed for serum and each organ was homogenized. GroupsIII-VIII were sacrificed at 2, 4, 8, 12, 18 and 24 hours followingdosing, respectively, and the same samples were collected. Group IX wastreated topically with 0.1 g of the test article for seven days (Days0-6). On day 7, 24 hours following the final application of the testarticle on Day 6, groups I and IX were sacrificed and the same samplescollected as for the previous groups. Each sample was measured fordisintegrations per minute (DPM) and the mean DPM/sample type wascalculated for each group.

Evaluation was based on the measurement of levels of radioactivity ofserum, urine, feces and target organs (liver, pancreas and spleen) atthe various time points with the objective of determining relativelevels of percutaneous penetration of the test article over 24 hours anddetermining where the drug accumulates with the method of application.

A summary of the average sample weights in grams for each group ispresented in the chart below.

TABLE 62 Pancreas Liver Spleen Feces Urine Blood Group I 0.2907 1.44680.0776 0.0654 NA 0.4318 Group II 0.1691 1.3352 0.0935 0.0164 NA 0.4530Group III 0.1300 1.0688 0.1777 0.0324 0.0890 0.4429 Group IV 0.13770.9893 0.0846 0.0292 0.0802 0.3770 Group V 0.1780 0.7105 0.0760 0.02990.0864 0.3222 Group VI 0.1156 0.8994 0.0595 0.0328 NA 0.3273 Group VII0.2864 1.1312 0.3355 0.0160 0.0671 0.2077 Group VIII 0.1969 1.19290.0905 0.0350 0.0097 0.3093 Group IX 0.3068 1.2912 0.0839 0.1034 NA0.3439 Mean 0.2012 1.1184 0.1199 0.041 0.0665 0.3572

Disintegrations per minute (DPM) are presented in Table 63 and weremeasured on a scintillation counter for each type of tissue sample fromeach animal. The average DPM for each sample type was calculated. Afterthe results were converted to 1 mL amounts, the averages were thendivided by average organ weights to obtain the DPM per tissue gramresult. The control (Group I) results were then subtracted from each ofthe other group results to remove background radiation amounts andobtain the actual number of average DPM per tissue gram for each samplein the group. By dividing by a constant of 2,220,000, the results wereconverted to microcuries per tissue gram. The final results representpicocuries (microcuries×1000) per tissue gram. The results for theorgans were presented in the chart-below and in FIG. 41. Please notethat the present application does not contain a Table 64.

Target Organ Results

TABLE 63 Picocuries per tissue gram Pancreas Liver Spleen Hr 0 - GroupII 1.09 10.34 0.40 Hr 2 - Group III 0.87 0.14 0.09 Hr 4 - Group IV 0.471.11 1.07 Hr 8 - Group V 6.05 2.80 0.45 Hr 12 - Group VI 0.13 0.96 0.46Hr 18 - Group VII 0.03 2.02 −0.15 Hr 24 - Group VIII 0.07 2.40 0.30

The data reflects that the test article accumulated substantially in thepancreas at approximately eight (8) hours after dosing and also, inlesser amounts, in the liver at eight hours after dosing. The amount ofpicocuries per tissue gram in the spleen and pancreas decreased tonearly zero by 18 hours after dosing. The Hour 0 liver results wereabnormal because of a single animal with an exceptionally high amount ofDPM at zero hours after dosing. One possible explanation for thisabnormally high result is that the animal managed to ingest the testarticle directly, either by licking its own skin or licking its pawsafter rubbing the dose site. That possibility would send the testarticle to the liver much quicker than percutaneous absorption. Afterthe 8 hour peak, the liver amounts decreased slightly at 12 hours butthen increased slightly at 18 hours and remained consistent through 24hours.

Target Organ Results (Cont.)

Of the amount of test article that accumulated in each of the targetorgans for all animals in Groups II-VIII, the percentage of picocuriesper tissue grams is presented in the chart below.

TABLE 65 Pancreas Liver Spleen Hr 0 12.51% 52.30% 15.21% Hr 2 9.99%0.71% 3.42% Hr 4 5.40% 5.61% 40.68% Hr 8 69.46% 14.16% 17.49% Hr 121.49% 4.86% 17.49% Hr 18 0.34% 10.22% −5.70% Hr 24 0.80% 12.14% 11.41%

Groups II-VIII were dosed with 4.112 microcuries of test article. Thechart below presents the amount of microcuries in each target organ ateach time period.

TABLE 66 Pancreas Liver Spleen Group II - 0 hrs 0.0011 0.0103 0.0004Group III - 2 hrs 0.0009 0.0001 0.0001 Group IV - 4 hrs 0.0005 0.00110.0011 Group V - 8 hrs 0.0060 0.0028 0.0005 Group VI - 12 hrs 0.00010.0010 0.0005 Group VII - 18 hrs 0.0000 0.0020 −0.0002 Group VIII - 24hrs 0.0001 0.0024 0.0003

By dividing those numbers by 4.112 (the amount of microcuries in eachdose), a percentage results that represents the amount of test articlethat was in each organ for each time period. The chart below presentsthose percentages.

TABLE 67 Pancreas Liver Spleen Hr 0 0.03% 0.25% 0.01% Hr 2 0.02% 0.00%0.00% Hr 4 0.01% 0.03% 0.03% Hr 8 0.15% 0.07% 0.01% Hr 12 0.00% 0.02%0.01% Hr 18 0.00% 0.05% 0.00% Hr 24 0.00% 0.06% 0.01%

The average percentage of test article in microcuries that reached thetarget organs is 0.03%, 0.07% and 0.01% for the pancreas, liver andspleen, respectively.

For the body waste samples, final results were presented in picocuriesper mL. This amount was obtained by converting the DPM to 1 mL,subtracting the Group I results, and then dividing by the constant2,220,000 to obtain microcuries per mL. By multiplying that result by1000, picocuries per mL was obtained. The average picocuries per mL forthe feces and the urine are presented in the chart below and in FIG. 42.Please note that the present application does not contain a Table 69.

TABLE 68 1 mL 1 mL Feces Urine Hr 0 - Group II −0.0433 0.0002 Hr 2 -Group III 18.2417 0.0000 Hr 4 - Group IV 3.9548 0.0305 Hr 8 - Group V117.1009 0.0081 Hr 12 - Group VI 52.7089 −0.0015 Hr 18 - Group VII0.7791 0.0057 Hr 24 - Group VIII 0.1303 0.0016

The data reflects that the test article accumulated substantially in thefeces at eight hours after dosing, continued to be present at 12 hoursafter dosing but was substantially lower by 18 hours after dosing. Therewas no indication that the test article accumulated in the urine at anypoint during the study.

Blood results were calculated in the same way as the waste sampleresults were calculated. The calculations for the picocuries per mL inthe blood resulted in negative numbers because of the high amount of DPMin Group I-Control's blood results. This was a result of two animalshaving higher than expected DPM readings in the blood. The results forthe blood are presented in the chart below and in FIG. 43. Please notethat the present application does not contain a Table 71.

TABLE 70 1 mL Blood Hr 0 - Group II −0.3706 Hr 2 - Group III −0.3896 Hr4 - Group IV −0.2877 Hr 6 - Group V −0.0890 Hr 12 - Group VI −0.1965 Hr18 - Group VII 0.2164 Hr 24 - Group VIII −0.0545

The data indicates that the test article did not accumulate in theblood; with the exception that test article may have been present in theblood at 18 hours after dosing. It is an oddity that there was nosignificant amounts of test article found in the blood, especially sincetest article was observed in the liver and pancreas. One explanation ispercutaneous absorption of the test article through the skin directly tothe organs; however, it is unlikely that the test article would notenter the blood after penetrating the skin. Another possibility is thatthe blood immediately recognized a foreign substance and deposited thetest material in the liver. The last dosing for Group II was at 10:23 amand the first sacrificed for Group II was at 10:45 am. Twenty-twominutes may have been enough time for the blood to rid itself of theforeign material.

Group IX data was calculated separately as the animals in Group IX weredosed repeatedly instead of once and were allowed 7 days to absorb thetest article. Group IX data is presented in the charts below.

TABLE 72 Picocuries per tissue gram for organ samples Pancreas LiverSpleen Group IX 0.12 2.07 1.19

Average picocuries per tissue gram for Groups II-VIII were 1.24, 2.83and 0.38 for the pancreas, liver and spleen respectively. Group IXresults for the organs were lower than average for the pancreas, closeto average for the liver and higher than average for the spleen. Thedata indicates that there were increasing amounts of test article in thespleen by Day 7 and that the amounts of test article in the liver at Day7 were comparable to the same amounts found in the liver at 18 and 24hours after dosing.

TABLE 73 Picocuries per mL for waste samples Feces Urine Group IX25.0650 −0.0011

Average picocuries per mL for the feces for Groups II-VIII was 27.55 andfor urine was 0.0064. Group IX feces and urine results were not abnormaland showed only minimal signs of test article in the feces.

TABLE 74 Picocuries per mL for blood Blood Group IX −0.0538

As with other groups, Group IX blood results indicate that the testarticle was not present in the blood.

The overall results indicate that there were no significant differenceswithin the weights of the target organs, feces, urine or blood amountsbetween groups. No significant amounts of the test article were detectedin the urine. There was no urine collected from Groups I (control), II(Hour 0) or VI (Hour 12). Except for Group VII (18 hours after dosing),the test article was not detected in significant amounts in the blood atany time during the study. The highest amounts of picocuries per tissuegram and picocuries per mL were recorded for Group V (8 hours afterdosing) and were found most concentrated in the feces and the pancreasat that time. Also in Group V, increased levels of picocuries per tissuegram were found in the liver. Within the liver, after a slight dip atHour 12, levels remained consistent at Hour 18 and Hour 24 andcomparable levels were found in the liver for Group IX (Day 7 animals).After the peak at 8 hours after dosing, the pancreas amounts dropped tonear zero levels, including Group IX results. The spleen amounts rose atHour 4 and then decreased to near zero levels by Hour 18. However thoseamounts increased for Group IX, indicating accumulated test article inthe spleen on Day 7. There was percutaneous absorption of the testarticle or a metabolite of the test material because the compound wasfound in the liver and the pancreas. It is strange that no test materialwas present in the blood in significant amounts since the expected routeof transport would be the blood flow. A possible explanation could berapid Clearance of the test article from the blood by the liver andpancreas. Another possibility is that the test article could have beeningested directly by the animals, either by licking the dose itself orlicking paws that had rubbed on the dose site.

Example 42 Western Analysis of Cells Treated with Coenzyme Q10

Over the past five decades enormous volume of information has beengenerated implicating endogenous/exogenous factors influencing specificprocesses as the underlying cause of malignant transformations. Clinicaland basic literature provides evidence that changes in the DNA structureand function play a significant role in the initiation and progressionof cancer, defining cancer as a genetic disease (Wooster, 2010; Haiman,2010). In the early 1920s, Otto Warburg and other investigators involvedin characterizing fundamental changes in etiology of oncogenesisdescribed two major observations (a) the ability of cells to transportand utilize glucose in the generation of ATP for energy production inthe presence of oxygen—also known as Warburg Effect and (b) alterationsin the mitochondrial structure and function—including changes in theelectron transport leading to a decrease in the production ofmitochondrial ATP. The past few years has seen a resurgence in theinvestigating the central role of cellular bioenergetics in the etiologyof cancer i.e. viewing cancer as a metabolic disease.

Historically, although mutations in genes has been thought to beresponsible for changes in gene expression, there is accumulatingliterature in support of epigenetic processes playing a critical role ininfluencing gene expression in supporting carcinogenesis. This isevidenced by the observation that mutation rate for most genes is lowand cannot account for the numerous (spectrum of) mutations found in thecancer cells. Epigenetic alteration is regulated by methylation andmodification of histone tails, both changes inherently linked to theenergy (nutrient) status of the cells since they require theavailability of co-factors e.g. acetyl CoA requirement for histoneacetylation (ref). The biosynthesis of acetyl CoA depends on glycolysisand Kreb's Cycle, directly linking the intracellular energy status toregulation of gene expression and activity.

In normal cells, mitochondrial oxidative phosphorylation generatessufficient ATP to meet the energy demands for maintaining normalphysiological activities and cell survival. A consequence ofmitochondrial energy production is the generation of reactive oxygenspecies (ROS), aberrant production of which leads to damage ofmitochondria (refs). It is well established that chronic ROS generationby the mitochondria leads to cumulative accumulation of geneticmutations, a phenomenon that has been implicated in the etiology ofcarcinogenesis. It has been suggested that cancer cells decreasemitochondrial respiration to minimize ROS generation, and switch toglycolysis to sustain energy production. Thus, a progressive shift ofenergy generation from oxidative phosphorylation to glycolysis would beessential for a cell to maintain energy production to maintainphysiological functions and could be associated with the progression ofa normal cell phenotype to that of a cancer cell. The progressive shiftin cellular energy (bioenergetic) profile in tandem with accumulatedalteration (mutations) in mitochondrial genetic make-up alters thecellular metabolome. Changes in the whole cell metabolomic profile as aconsequence of mitochondrial phosphorylation to glycolysis transitioncorresponds to an abnormal bioenergetic induced metabolomic profile andis the underlying cause supporting carcinogenesis. Targeted interventionusing an endogenous molecule to elicit a cellular metabolomic shifttowards conditions of a non-cancerous normal mitochondrial oxidativephosphorylation associated cellular bioenergetic state represents atherapeutic endpoint in the treatment of cancer.

Coenzyme Q10 as a MIM Causing an Epi-Metabolomic Shift

The data presented herein demonstrates that treatment of normal andcancer cells with Coenzyme Q10 is associated with changes in theexpression of proteins that regulate key biochemical terminals withinthe glycolysis—mitochondrial oxidative stress continuum. The combinationof data describing assessment of protein expression by western blottingand oxygen consumption rates demonstrates that in normal cells, there isno significant alteration in normal glycolytic and mitochondrialrespiration rates following exposure to Coenzyme Q10. Thus, the valuesfor expression of the proteins and mitochondrial respiration rates innormal cell lines e.g. HDFa (normal human adult fibroblast), HASMC(normal human aortic smooth muscle cell), nFib (normal fibroblast) andHeKa (normal human keratinocytes) can be considered as representativesof baseline physiological state. Any deviation in expression of proteinsand mitochondrial respiration rates in cancer cell lines, e.g. HepG2(liver cancer), PaCa-2 (pancreatic cancer), MCF7 (breast cancer), SK-MEL(melanoma) and SCC-25 (squamous cell carcinoma), is representative ofalteration due to initiation/progression of the disease, in this casecancer. The experimental evidence provides support to the hypothesisthat exposure of Coenzyme Q10 to cancer cells is associated withcellular pathophysiological reorganization that is reminiscent of normalcells. Specifically, the data provided herein demonstrates that CoenzymeQ10 exposure in cancer cells is associated with a shift in theglycolytic pathways and mitochondrial oxidative phosphorylationresponsible for induction of global reorganization of cellulararchitecture to that observed in normal cells.

In normal cells, the end-points of glycolytic output are linked tomitochondrial oxidative phosphorylation (OXPHOS), i.e. generation ofpyruvate from glucose via the glycolytic pathway for the entry into theKreb's Cycle (also known as Tricarboxylic acid cycle, TCA, or CitricAcid Cycle) to generate reducing equivalents to support themitochondrial OXPHOS for ATP production. Thus, in normal cells theexpression and functional orientation of gene products involved inglycolysis is primed towards adequate generation of pyruvate and itsentry into the Kreb's Cycle. Dysregulated expression and function of keyproteins participating in glycolysis and Kreb's Cycle pathways in cancercells results in enhanced glycolysis with a significant decrease inmitochondrial function. Exposure of cancer cells to Coenzyme Q10, anendogenous molecule that selectively influences the mitochondrialrespiratory chain, alters (normalizes) expression of proteins of theglycolyis and Kreb's Cycle pathways to facilitate a bioenergetic shiftsuch that energy production (i.e. ATP generation) is restored to themitochondria.

EXPERIMENTAL PROCEDURE Western Blot Experiment 1

The cells that were used for the experiment were HDFa, and MCF-7 cellsthat were treated or not with Coenzyme Q10 at two differentconcentrations, 50 μM and 100 μM, and harvested after 24 hours oftreatment. The whole cell pellets were resuspended one at a time in 1 mLof C7 buffer and transferred to labeled 15 mL tubes. The samples werethen sonicated in the cold room on ice using 6 sonic pulses with thesetting at #14. The samples were spun for a short time to 2500 g aftersonication and the samples transferred to 2 ml tubes. The pH wasverified of each sample (pH should be 9.0) using the foam remaining inthe 50 mL sample tubes.

Alkylation and reduction of samples was performed for each sample byadding 10 ul of 1M acrylamide, 25 ul of tributylphoshene and incubationfor 90 mins with intermittent mixing. After incubation, 10 ul of 1M DTTwas added and the tubes were spun at 20,000 g at 20 deg C. for 10minutes and transferred the supernatant to labeled Amicon Ultracentrifugal filter units with a 10 k cut off (Millipore catalog #UFC801024). The samples were spun for 15 minutes at 2500 g in 2 intervals.The conductivity was measured for Chaps alone as well as the samplesusing a conductivity meter. If the conductivity of samples is high, then1 ml of chaps was added for buffer exchange and spun again at 2500 guntil the volume was down to 250 ul. When the conductivity was 200 orless the samples were spun in 5 min intervals at 2500 g until the volumeof the supernatant was between 150-100 ul. The sample supernatants weretransferred to eppendorf tubes and Bradford assay was performed usingBSA as standard.

The samples were processed as per standard protocols as described aboveand the amount of protein in each of the samples was determined byBradford assay. Sample volumes equivalent to 10 ug of protein wereprepared as shown below with Lamelli Loading dye (LDS) and MilliQ waterwere run on a 4-12% Bis-Tris Novex NuPAGE gel (Invitrogen, Cat#NP0323Box)

The gels were run for 50 minutes using 1X MOPS buffer using a NOVEXXcell Surelock system at 200 V. The gels were then transferred for 1hour using a NOVEX Xcell Surelock wet transfer protocol at 30 V. Theblots were stained with Simply Blue Safestain from Invitrogen (LC6065).

IDH1 and ATP Citrate Lyase Levels in HDFa and MCF-7 Samples.

After transfer each of the blots was placed in between 2 Whatman Filterpapers and dried for 15-20 minutes. After drying the blots were labeledwith the date, the type of samples and either blot 1 or blot 2 using aHB pencil. The molecular weight markers were outlined with the penciland with single lines for the blue and a doublet for the coloredmarkers. The blots were activated with methanol for 5 seconds, washedwith water for 5 minutes, and TBST for 15 minutes. The blots wereblocked for 1 hour with 5% blocking reagent in TBS-T at room temperatureand then washed 3 times with TBS-T (1X−15′; 2X5′ each). Blot 1 wasprobed with the primary antibody for IDH1 (Cell Signaling #3997) in TBSTwith 5% BSA (at 1:1000 dilutions) and blot 2 with the rabbit polyclonalantibody for ATP Citrate Lyase in 5% BSA (Cell Signaling #4332) at1:1000 dilution by incubation overnight at 4 deg C. with shaking. Afterthe overnight incubation with primary antibodies, the blots were washed3 times with TBS-T (1X−15′; 2X5′ each) and probed with the secondaryantibody (antirabbit; 1:10,000 dilution) for 1 h on the orbital tiltingshaker at room temperature. After 1 h of incubation with secondaryantibodies, the blots were washed 3 times with TBS-T (1X−15′; 2X5′ each)and then incubated with ECF reagent for 5 mins and then each blotscanned with 5100 Fuji Laser scanner at 25 uM resolution, 16 bit, greenlaser, at 400V and at 500 V.

Actin Levels in HDFa and MCF-7 Samples.

The above blots were stripped by incubating for 30 minutes withmethanol, followed by two 10 minute washes with TBS-T, then 30 minutesof incubation with Stripping buffer at 50 deg C., and followed by twowashes with 100 ml or more of TBS-T for 30′ each. The 2 blots werescanned in laser scanner to check for complete stripping. The blots werethen activated with methanol for 5 seconds, washed with water for 5minutes, and TBST for 15 minutes. The blots were blocked for 1 hour with5% blocking reagent in TBS-T at room temperature and then washed 3 timeswith TBS-T (1X−15′; 2X5′ each) and probed with the antibody for Actin in5% BSA (Sigma catalog #A5316, clone AC-74) at 1:5000 dilutions for 1hour at room temperature with shaking. After 1 hour of incubation withprimary antibody for Actin, the membranes were washed 3 times with TBS-T(1X−15′; 2X5′ each) and probed with the secondary antibody (antimouse;1:10,000 dilution) for 1 h on the orbital tilting shaker at roomtemperature. After 1 h of incubation with secondary antibodies, theblots were washed 3 times with TBS-T (1X−15′; 2X5′ each) and thenincubated with ECF reagent for 5 minutes and then each blot scanned with5100 Fuji Laser scanner at 25 uM resolution, 16 bit, green laser, at400V and at 500 V.

Western Blot Experiment 2

The cells used in this experiment were SKMEL28, SCC-25, nFib and Hekathat were treated or not with coenzyme Q10 at two differentconcentrations, 50 μM or 100 1.1M, and harvested after 3, 6 and/or 24hours of treatment. The samples were processed and run on a 4-12%Bis-Tris Novex NuPAGE gel as described above. The gels were run,transferred and stained essentially as described above.

Levels of IDH1 for the 4 Cell Lines

After transfer the blot was dried for 15-20 minutes, activated withmethanol for 5 seconds, washed with water for 5 minutes, and TBST for 15minutes. The blot was blocked for 1 hour with 5% blocking reagent inTBS-T at room temperature and then washed 3 times with TBS-T (1X−15′;2X5′ each). This was then probed with the primary antibody for IDH1(Cell Signaling #3997) in TBST with 5% BSA (at 1:1000 dilutions) byincubation overnight at 4 deg C. with shaking. After the overnightincubation with primary antibody for IDH1, the blot was washed 3 timeswith TBS-T (1X−15′; 2X5′ each) and probed with the secondary antibody(antirabbit; 1:10,000 dilution) for 1 h at room temperature. After 1 hof incubation with secondary antibodies, the blot was washed 3 timeswith TBS-T (1X−15′; 2X5′ each) and then incubated with ECF reagent for 5mins and then each blot scanned with 5100 Fuji Laser scanner at 25 uMresolution, 16 bit, green laser, at 400V and at 500 V.

ATP Citrate Lyase Levels in 4 Different Cell Lines.

The Isocitrate dehydrogenase blot was stripped by incubating for 30minutes with methanol, followed by two 10 minute washes with TBS-T, then30 minutes of incubation with stripping buffer at 50 deg C., andfollowed by two washes with 100 ml or more of TBS-T for 30′ each. Theblot was scanned in laser scanner to check for complete stripping. Theblot was activated with methanol for 5 seconds, washed with water for 5minutes, and TBST for 15 minutes. The blot was blocked for 1 hour with5% blocking reagent in TBS-T at room temperature and then washed 3 timeswith TBS-T (1X−15′; 2X5′ each). This was then probed with the rabbitpolyclonal antibody for ATP Citrate Lyase in 5% BSA (Cell Signaling#4332) at 1:1000 dilution overnight at 4 deg C. with shaking. After theovernight incubation with primary antibody for ATP Citrate Lyase, themembrane was washed 3 times with TBS-T (1X−15′; 2X5′ each) and probedwith the secondary antibody (antirabbit; 1:10,000 dilution) for 1 h onthe orbital tilting shaker at room temperature. After 1 h of incubationwith secondary antibodies, the blot was washed 3 times with TBS-T(1X−15′; 2X5′ each) and then incubated with ECF reagent for 5 minutesand then each blot scanned with 5100 Fuji Laser scanner at 25 uMresolution, 16 bit, green laser, at 400V and at 500 V.

Actin Levels in 4 Different Cell Lines.

The ATP Citrate Lyase blot was stripped by incubating for 30 minuteswith methanol, followed by two 10 minute washes with TBS-T, then 30minutes of incubation with Stripping buffer at 50 deg C., and followedby two washes with 100 ml or more of TBS-T for 30′ each. The blot wasscanned in laser scanner to check for complete stripping. The blot wasactivated with methanol for 5 seconds, washed with water for 5 minutes,and TBST for 15 minutes. The blot was blocked for 1 hour with 5%blocking reagent in TBS-T at room temperature and then washed 3 timeswith TBS-T (1X−15′; 2X5′ each) and probed with the antibody for Actin in5% BSA (Sigma catalog #A5316, clone AC-74) at 1:5000 dilutions for 1hour at room temperature with shaking. After 1 hour of incubation withprimary antibody for Actin, the membranes were washed 3 times with TBS-T(1X−15′; 2X5′ each) and probed with the secondary antibody (antimouse;1:10,000 dilution) for 1 h on the orbital tilting shaker at roomtemperature. After 1 h of incubation with secondary antibodies, theblots were washed 3 times with TBS-T (1X−15′; 2X5′ each) and thenincubated with ECF reagent for 5 minutes and then each blot scanned with5100 Fuji Laser scanner at 25 uM resolution, 16 bit, green laser, at400V and at 500 V.

Western Blot Experiment 3

The cells used in this experiment were HepG2, HASMC, and PACA2 cellsthat were treated or not with Coenzyme Q10 at two differentconcentrations (50 μM and 100 1.1M) and harvested 48 hours of treatment.In this experiment (western blot experiment 3), and in all of theexperiments described below in this Example (i.e., western blotexperiments 4 through 9), the cells were additionally treated witheither 5 mM glucose (“5G”) or 22 mM glucose (“22G”). The samples derivedfrom the cells were processed and run on a 4-12% Bis-Tris Novex NuPAGEgel as described above. The gels were run, transferred and stainedessentially as described above.

IDH1, ATP Citrate Lyase and Actin Levels in HASMC vs. PACA2 and HepG2.

The levels of IDH1, ATP citrate lyase and actin levels were determinedby probing the blots with primary antibodies for IDH1, ATP citrate lyaseand actin, essentially as described above.

Western Blot Experiment 4

The cells used in this experiment were HepG2 cells that were treated ornot with Coenzyme Q10 at two different concentrations, 50 or 100 μM, andharvested after 24 or 48 hours of treatment. The samples were processedand run on a 4-12% Bis-Tris Novex NuPAGE gel as described above. Thegels were run, transferred and stained essentially as described above.

Lactate Dehydrogenase Levels in HepG2 Cells.

After transfer each blot was dried for 15-20 minutes, activated withmethanol for 5 seconds, washed with water for 5 minutes, and TBST for 15minutes. The blots were blocked for 1 hour with 5% blocking reagent inTBS-T at room temperature and then washed 3 times with TBS-T (1X−15′;2X5′ each) and probed with the primary antibody for LactateDehydrogenase (abcam ab2101; polyclonal) in 5% BSA (at 1:1000 dilutions)by incubation overnight at 4 deg C. with shaking. After the overnightincubation with primary antibody for Lactate Dehydrogenase, the blotswere washed 3 times with TBS-T (1X−15′; 2X5′ each) and probed with thesecondary antibody (rabbit antigoat; 1:10,000 dilution) for 1 h at roomtemperature. After 1 h of incubation with secondary antibodies, theblots were washed 3 times with TBS-T (1X−15′; 2X5′ each) and thenincubated with ECF reagent for 5 mins and then each blot scanned with5100 Fuji Laser scanner at 25 uM resolution, 16 bit, green laser, at400V and at 500 V.

Pyruvate Kinase Muscle form (PKM2) Levels in HepG2 Cells.

The lactate dehydrogenase blots were stripped by incubating for 30minutes with methanol, followed by two 10 minute washes with TBS-T, then30 minutes of incubation with Stripping buffer at 50 deg C., andfollowed by two washes with 100 ml or more of TBS-T for 30′ each. The 2blots were scanned in laser scanner to check for complete stripping. Theblots were activated with methanol for 5 seconds, washed with water for5 minutes, and TBST for 15 minutes. The blots were blocked for 1 hourwith 5% blocking reagent in TBS-T at room temperature and then washed 3times with TBS-T (1X−15′; 2X5′ each) and probed with the rabbitpolyclonal antibody for Pyruvate Kinase M2 in 5% BSA (NOVUS BIOLOGICALScatalog #H00005315-D01P) at 1:500 dilution overnight at 4 deg C. withshaking. After the overnight incubation with primary antibody forPyruvate Kinase M2, the membranes were washed 3 times with TBS-T(1X−15′; 2X5′ each) and probed with the secondary antibody (antirabbit;1:10,000 dilution) for 1 h on the orbital tilting shaker at roomtemperature. After 1 h of incubation with secondary antibodies, theblots were washed 3 times with TBS-T (1X-15′; 2X5′ each) and thenincubated with ECF reagent for 5 minutes and then each blot scanned with5100 Fuji Laser scanner at 25 uM resolution, 16 bit, green laser, at400V and at 500 V.

Pyruvate Dehydrogenase Beta Levels in HepG2 Cells.

The pyruvate kinase blots were stripped by incubating for 30 minuteswith methanol, followed by two 10 minute washes with TBS-T, then 30minutes of incubation with Stripping buffer at 50 deg C., and followedby two washes with 100 ml or more of TBS-T for 30′ each. The 2 blotswere scanned in laser scanner to check for complete stripping. Aftermaking sure stripping of the antibody and the ECF reagent has worked,the blots were activated with methanol for 5 seconds, washed with waterfor 5 minutes, and TBST for 15 minutes. The blots are blocked for 1 hourwith 5% blocking reagent in TBS-T at room temperature and then washed 3times with TBS-T (1X−15′; 2X5′ each) and probed with the antibody forPyruvate Dehydrogenase in 5% BSA (ABNOVA catalog #H00005162-M03) at1:500 dilutions) overnight at 4 deg C. with shaking. After the overnightincubation with primary antibody for Pyruvate Dehydrogenase, themembranes were washed 3 times with TBS-T (1X−15′; 2X5′ each) and probedwith the secondary antibody (antimouse; 1:10,000 dilution) for 1 h onthe orbital tilting shaker at room temperature. After 1 h of incubationwith secondary antibodies, the blots were washed 3 times with TBS-T(1X−15′; 2X5′ each) and then incubated with ECF reagent for 5 minutesand then each blot scanned with 5100 Fuji Laser scanner at 25 uMresolution, 16 bit, green laser, at 400V and at 500 V.

Actin Levels in HepG2 Cells.

The Pyruvate Dehydrogenase blots were stripped and then reprobed foractin, essentially as described above.

Western Blot Experiment 5

The cells used in this experiment were MIAPACA2 (PACA2) cells that weretreated or not with Coenzyme Q10 at two different concentrations, 50 or100 μM, and harvested after 24 or 48 hours of treatment. The PACA2samples were processed and the gels were run, transferred, stained andscanned essentially as described above.

Lactate Dehydrogenase (LDH) and Pyruvate Dehydrogenase (PDH) Levels inPaCa2 Cells

The levels of LDH and PDH were determined by probing the blotssuccessively with primary antibodies for LDH and PDH, essentially asdescribed above.

Caspase 3 Levels in PaCa2 Cells.

The blots were stripped by incubating for 30 minutes with methanol,followed by two 10 minute washes with TBS-T, then 30 minutes ofincubation with Stripping buffer at 50 deg C., and followed by twowashes with 100 ml or more of TBS-T for 30′ each. The 2 blots werescanned in laser scanner to check for complete stripping. The blots wereactivated with methanol for 5 seconds, washed with water for 5 minutes,and TBST for 15 minutes. The blots were blocked for 1 hour with 5%blocking reagent in TBS-T at room temperature and then washed 3 timeswith TBS-T (1X−15′; 2X5′ each) and probed with the antibody for Caspase3 in 5% BSA (Santacruz Biotechnology #sc7272) at 1:200 dilutions)overnight at 4 deg C. with shaking. After the overnight incubation withprimary antibody for Caspase 3, the membranes were washed 3 times withTBS-T (1X-15′; 2X5′ each) and probed with the secondary antibody(antimouse; 1:10,000 dilution) for 1 h on the orbital tilting shaker atroom temperature. After 1 h of incubation with secondary antibodies, theblots were washed 3 times with TBS-T (1X−15′; 2X5′ each) and thenincubated with ECF reagent for 5 minutes and then each blot scanned with5100 Fuji Laser scanner at 25 uM resolution, 16 bit, green laser, at400V and at 500 V.

Western Blot Experiment 6

The cells that were used for this Western blot experiment were PC-3,HepG2, MCF-7, HDFa and PACA2 that were treated or not with a CoenzymeQ10 IV formulation and harvested after 24 hours of treatment. Thesamples were processed and the gels were run, transferred, stained andscanned essentially as described above. Capase 3 and Actin levels indifferent cell types.

The levels of Caspase 3 and actin were determined by probing the blotssuccessively with primary antibodies for Caspase 3 and actin,essentially as described above.

Western Blot Experiment 7

The cells used in this experiment were Human Aortic Smooth Muscle(HASMC) cells that were treated or not with Coenzyme Q10 at twodifferent concentrations, 50 μM or 100 μM, and harvested after 24 or 48hours of treatment. The HASMC samples were processed and the gels wererun, transferred, stained and scanned essentially as described above.

Experimental Protocol for Actin:

The levels of actin were determined by probing the blots with a primaryantibody for actin, essentially as described above.

Experimental Protocol for Hif Lalplia, Caspase3 and PDHB:

The Actin blots were stripped by incubating for 30 minutes withmethanol, followed by two 10 minute washes with TBS-T, then 30 minutesof incubation with Stripping buffer at 50 deg C., and followed by twowashes with 100 ml or more of TBS-T for 30′ each. The blots were scannedin laser scanner to check for complete stripping. The blots wereactivated with methanol for 5 seconds, washed with water for 5 minutes,and TBST for 15 minutes. The blots were blocked for 1 hour with 5%blocking reagent in TBS-T at room temperature and then washed 3 timeswith TBS-T (1X−15′; 2X5′ each) and probed with the primary antibody forHif 1 alpha, Caspase 3 or PDHB in 5% BSA (at 1:200 by incubationovernight at 4 deg C. with gentle shaking. The primary antibody for Hif1 alpha (Abcam ab2185; antirabbit) was at 1:500 dilution in 5% BSA. Theprimary antibody for Caspase 3 (Santacruz sc7272; antirabbit) was at1:200 dilution in 5% BSA. The primary antibody for PyruvateDehydrogenase beta (PDHB) (Novus Biologicals H00005162-M03; antimouse)was at 1:500 dilution in 5% BSA. After incubation with primaryantibodies, the membranes were washed 3 times with TBS-T (1X−15′; 2X5′each) and probed with the secondary antibody (PDHB antimouse; Hif 1a andCaspase 3 antirabbit; 1:10,000 dilution) for 1 h at room temperature.After 1 h of incubation with secondary antibodies, the blots were washed3 times with TBS-T (1X-15′; 2X5′ each) and then incubated with ECFreagent for 5 minutes and then each blot scanned with 5100 Fuji Laserscanner at 25 uM resolution, 16 bit, green laser, at 400V and at 500 V.

Experimental Protocol for PKM2, SDHB and SDHC:

The above blots were stripped by incubating for 30 minutes withmethanol, followed by two 10 minute washes with TBS-T, then 30 minutesof incubation with Stripping buffer at 50 deg C., and followed by twowashes with 100 ml or more of TBS-T for 30′ each. The blots were scannedin laser scanner to check for complete stripping. The blots wereactivated with methanol for 5 seconds, washed with water for 5 minutes,and TBST for 15 minutes. The blots were blocked for 1 hour with 5%blocking reagent in TBS-T at room temperature and then washed 3 timeswith TBS-T (1X−15′; 2X5′ each) and probed with the primary antibody forPKM2, SDHB or SDHC in 5% BSA in TBS-T by incubation overnight at 4 degC. with gentle shaking. The primary antibody for SDHC (ABNOVAH00006391-M12; antimouse) was at 1:500 dilution. The primary antibodyfor SDHB was from Abcam ab4714-200; antimouse; at 1:1000 dilution. Theprimary antibody for Pyruvate Kinase M2 (PKM2) was from NovusBiologicals H00005315-D0IP; antirabbit; at 1:500 dilution. Afterincubation with primary antibodies, the membranes were washed 3 timeswith TBS-T (1X−15′; 2X5′ each) and probed with the secondary antibody(SDHB & C antimouse; and PKM2 antirabbit; 1:10,000 dilution) for 1 h onthe orbital tilting shaker at room temperature. After 1 h of incubation,the blots were washed 3 times with TBS-T (1X−15′; 2X5′ each) andincubated with ECF reagent for 5 minutes and then each blot scanned with5100 Fuji Laser scanner at 25 uM resolution, 16 bit, green laser, at400V and at 500 V.

Experimental Protocol for LDH and Bik:

The above blots were stripped by incubating for 30 minutes withmethanol, followed by two 10 minute washes with TBS-T, then 30 minutesof incubation with Stripping buffer at 50 deg C., and followed by twowashes with 100 ml or more of TBS-T for 30′ each. The blots were scannedin laser scanner to check for complete stripping. The blots wereactivated with methanol for 5 seconds, washed with water for 5 minutes,and TBST for 15 minutes. The blots were blocked for 1 hour with 5%blocking reagent in TBS-T at room temperature and then washed 3 timeswith TBS-T (1X−15′; 2X5′ each) and probed with the primary antibody forLDH or Bik in 5% BSA in TBS-T by incubation overnight at 4 deg C. withgentle shaking. The primary antibody for LDH was from Abcam ab2101;antigoat; at 1:1000 dilution. The primary antibody for Bik was from CellSignaling #9942; antirabbit; at 1:1000 dilution. After incubation withprimary antibodies, the membranes were washed 3 times with TBS-T(1X−15′; 2X5′ each) and probed with the secondary antibody (LDHantigoat; Jackson Laboratories) and Bik antirabbit; 1:10,000 dilution)for 1 h on the orbital tilting shaker at room temperature. After 1 h ofincubation, the blots were washed 3 times with TBS-T (1X-15′; 2X5′ each)and incubated with ECF reagent for 5 minutes and then each blot scannedwith 5100 Fuji Laser scanner at 25 uM resolution, 16 bit, green laser,at 400V and at 500 V.

Western Blot Experiment 9

The cells used were HepG2 cells that were treated or not with CoenzymeQ10 at two different concentrations, 50 μM or 100 μM, and harvestedafter 24 or 48 hours of treatment. The HepG2 samples processed and thegels were run, transferred, stained and scanned essentially as describedabove.

Experimental Protocol for Actin:

The levels of actin were determined by probing the blots with a primaryantibody for actin, essentially as described above.

Experimental Protocol for Caspase3 and MMP-6:

The Actin blots were stripped by incubating for 30 minutes withmethanol, followed by two 10 minute washes with TBS-T, then 30 minutesof incubation with Stripping buffer at 50 deg C., and followed by twowashes with 100 ml or more of TBS-T for 30′ each. The blots wereactivated with methanol for 5 seconds, washed with water for 5 minutes,and TBST for 15 minutes. The blots were blocked for 1 hour with 5%blocking reagent in TBS-T at room temperature and then washed 3 timeswith TBS-T (1X−15′; 2X5′ each) and probed with the primary antibody forCaspase 3 or MMP-6 in 5% BSA by incubation overnight at 4 deg C. withgentle shaking. The primary antibody for Caspase 3 (Abcam ab44976-100;antirabbit) was at 1:500 dilution in 5% BSA. The primary antibody forMMP-6 (Santacruz scMM0029-ZB5; antimouse) was at 1:100 dilution in 5%BSA. After incubation with primary antibodies, the membranes were washed3 times with TBS-T (1X−15′; 2X5′ each) and probed with the secondaryantibody (MMP-6 antimouse; Caspase 3 antirabbit; 1:10,000 dilution) for1 h at room temperature. After 1 h of incubation with secondaryantibodies, the blots were washed 3 times with TBS-T (1X−15′; 2X5′ each)and then incubated with ECF reagent for 5 minutes and then each blotscanned with 5100 Fuji Laser scanner at 25 uM resolution, 16 bit, greenlaser, at 400V and at 500 V.

Experimental Protocol for LDH:

The above blots were stripped by incubating for 30 minutes withmethanol, followed by two 10 minute washes with TBS-T, then 30 minutesof incubation with stripping buffer at 50 deg C., and followed by twowashes with 100 ml or more of TBS-T for 30′ each. The blots wereactivated with methanol for 5 seconds, washed with water for 5 minutes,and TBST for 15 minutes. The blots ere blocked for 1 hour with 5%blocking reagent in TBS-T at room temperature and then washed 3 timeswith TBS-T (1X−15′; 2X5′ each) and probed with the primary antibody forLDH in 5% BSA or 5% milk by incubation overnight at 4 deg C. with gentleshaking. The primary antibody for LDH 080309b1 (Abcam ab2101; antigoat)was at 1:1000 dilution in 5% BSA. The primary antibody for LDH 080309b2(Abcam ab2101; antigoat) was at 1:1000 dilution in 5% milk. Afterincubation with primary antibodies, the membranes were washed 3 timeswith TBS-T (1X−15′; 2X5′ each) and probed with the secondary antibody(Jackson Immuno Research antigoat; 1:10,000 dilution; 305-055-045) for 1h. After 1 h of incubation with secondary antibodies, the blots werewashed 3 times with TBS-T (1X−15′; 2X5′ each) and then incubated withECF reagent for 5 minutes and then each blot scanned with 5100 FujiLaser scanner at 25 uM resolution, 16 bit, green laser, at 400V and at500 V.

Experimental Protocol for Transaldolase and Hifla:

The above blots were stripped by incubating for 30 minutes withmethanol, followed by two 10 minute washes with TBS-T, then 30 minutesof incubation with Stripping buffer at 50 deg C., and followed by twowashes with 100 ml or more of TBS-T for 30′ each. The blots wereactivated with methanol for 5 seconds, washed with water for 5 minutes,and TBST for 15 minutes. The blots are blocked for 1 hour with 5%blocking reagent in TBS-T at room temperature and then washed 3 timeswith TBS-T (1X−15′; 2X5′ each) and probed with the primary antibody forTransaldolase or Hif I a in 5% BSA by incubation overnight at 4 deg C.with gentle shaking. The primary antibody for Transaldolase (Abramab67467; antimouse) was at 1:500 dilution. The primary antibody forHif1a (Abram ab2185; antirabbit) was at 1:500 dilution. After incubationwith primary antibodies, the membranes were washed 3 times with TBS-T(1X−15′; 2X5′ each) and probed with the secondary antibody (antimouse orantirabbit; 1:10,000 dilution) for 1 h on the orbital tilting shaker atroom temperature. After 1 h of incubation with secondary antibodies, theblots were washed 3 times with TBS-T (1X-15′; 2X5′ each) and thenincubated with ECF reagent for 5 minutes and then each blot scanned with5100 Fuji Laser scanner at 25 uM resolution, 16 bit, green laser, at 400& 500V.

Experimental Protocol for IGFBP3 and TP53:

The above blots were stripped by incubating for 30 minutes withmethanol, followed by two 10 minute washes with TBS-T, then 30 minutesof incubation with Stripping buffer at 50 deg C., and followed by twowashes with 100 ml or more of TBS-T for 30′ each. The blots wereactivated with methanol for 5 seconds, washed with water for 5 minutes,and TBST for 15 minutes. The blots are blocked for 1 hour with 5%blocking reagent in TBS-T at room temperature and then washed 3 timeswith TBS-T (1X−15′; 2X5′ each) and probed with the primary antibody forIGFBP3 or TP53 in 5% BSA by incubation overnight at 4 deg C. with gentleshaking. The primary antibody for IGFBP3 (Abram ab76001; antirabbit) wasat 1:100 dilution. The primary antibody for TP53 (Sigma Aldrich AV02055;antirabbit) was at 1:100 dilution. After incubation with primaryantibodies, the membranes were washed 3 times with TBS-T (1X−15′; 2×5′each) and probed with the secondary antibody (antirabbit; 1:10,000dilution) for 1 h on the orbital tilting shaker at room temperature.After 1 h of incubation with secondary antibodies, the blots were washed3 times with TBS-T (1X−15′; 2X5′ each) and then incubated with ECFreagent for 5 minutes and then each blot scanned with 5100 Fuji Laserscanner at 25 uM resolution, 16 bit, green laser, at 400 & 500V.

Experimental Protocol for Transaldolase and PDHB:

The above blots were stripped by incubating for 30 minutes withmethanol, followed by two 10 minute washes with TBS-T, then 30 minutesof incubation with Stripping buffer at 50 deg C., and followed by twowashes with 100 ml or more of TBS-T for 30′ each. The blots wereactivated with methanol for 5 seconds, washed with water for 5 minutes,and TBST for 15 minutes. The blots were blocked for 1 hour with 5%blocking reagent in TBS-T at room temperature and then washed 3 timeswith TBS-T (1X−15′; 2X5′ each) and probed with the primary antibody forTransaldolase or PDHB in 5% BSA by incubation overnight at 4 deg C. withgentle shaking. The primary antibody for Transaldolase (Santacruzsc51440; antigoat) was at 1:200 dilution. The primary antibody for PDHB(Novus Biologicals H00005162-M03; antimouse) was at 1:500 dilution.After incubation with primary antibodies, the membranes were washed 3times with TBS-T (1X−15′; 2X5′ each) and probed with the secondaryantibody (antigoat or antimouse; 1:10,000 dilution) for 1 h on theorbital tilting shaker at room temperature. After 1 h of incubation withsecondary antibodies, the blots were washed 3 times with TBS-T (1X−15′;2X5′ each) and then incubated with ECF reagent for 5 minutes and theneach blot scanned with 5100 Fuji Laser scanner at 25 uM resolution, 16bit, green laser, at 400 & 500V.

Results Isocitrate Dehydrogenase-1 (IDH-1)

Isocitrate dehydrogenase is one of the enzymes that is part of the TCAcycle that usually occurs within the mitochondrial matrix. However, IDH1is the cytosolic form of the enzyme that catalyzes the oxidativedecarboxylation of isocitrate to α-ketoglutarate and generates carbondioxide in a two step process. IDH1 is the NADP⁺ dependent form that ispresent in the cytosol and peroxisome. IDH1 is inactivated by Ser113phosphorylation and is expressed in many species including those withouta citric acid cycle. IDH1 appears to function normally as a tumorsuppressor which upon inactivation contributes to tumorigenesis partlythrough activation of the HIF-1 pathway (Bayley 2010; Reitman, 2010).Recent studies have implicated an inactivating mutation in IDH1 in theetiology of glioblasotoma (Bleeker, 2009; Bleeker, 2010).

Treatment with Coenzyme Q10 increased expression of IDH1 in cancer celllines including MCF-7, SKMEL28, HepG2 and PaCa-2 cells. There was amoderate increase in expression in the SCC25 cell lines. In contrastcultures of primary human derived fibroblasts HDFa, nFIB and the humanaortic smooth muscle cells HASMC did not demonstrate significant changesin the expression pattern of the IDH1 in response to Coenzyme Q10.α-ketoglutarate (α-KG) is a key intermediate in the TCA cycle,biochemically synthesized from isocitrate and is eventually converted tosuccinyl coA and is a druggable MIM and EpiShifter. The generation ofα-KG serves as a critical juncture in the TCA cycle as it can be used bythe cell to replenish intermediates of the cycle, resulting ingeneration of reducing equivalents to increase oxidativephosphorylation. Thus, Coenzyme Q10 mediated increase in IDH1 expressionwould result in formation of intermediates that can be used by themitochondrial TCA cycle to augment oxidative phosphorylation in cancercells. The results are summarized in the tables below.

TABLE 75 IDH1 in HDFa and MCF-7 Composition Average Normalized IntensityHDF, Media 346 HDF24-50-Coenzyme Q10 519 HDF24-100-Coenzyme Q10 600 MCF,Media 221 MCF24-50-Coenzyme Q10 336 MCF24-100-Coenzyme Q10 649

TABLE 76 IDH1 in HASMC vs. HepG2 after Treatment Amount - CompositionNormalized Intensity HAS5g48-media 20 HAS5g48-50-Coenzyme Q10 948HAS5g48-100-Coenzyme Q10 1864 HAS22G48-Media 1917 HAS22G48-50-CoenzymeQ10 1370 HAS22G48-100-Coenzyme Q10 1023 Hep5g48-Media 14892Hep5g48-50-Coenzyme Q10 14106 Hep5g48-100-Coenzyme Q10 15774Hep22G48-Media 16558 Hep22G48-50-Coenzyme Q10 15537Hep22G48-100-Coenzyme Q10 27878

TABLE 77 IDH1 in HASMC vs. PACA2 after Treatment Amount - CompositionNormalized Intensity HAS5g48-media 562 HAS5g48-50-Coenzyme Q10 509HAS5g48-100-Coenzyme Q10 627 HAS22G48-Media 822 HAS22G48-50-Coenzyme Q101028 HAS22G48-100-Coenzyme Q10 1015 PACA5g48-Media 1095PACA5g48-50-Coenzyme Q10 1095 PACA5g48-100-Coenzyme Q10 860PACA22G48-Media 1103 PACA22G48-50-Coenzyme Q10 1503PACA22G48-100-Coenzyme Q10 1630

ATP Citrate Lyase (ACL)

ATP citrate Lyase (ACL) is a homotetramer (˜126 kd) enzyme thatcatalyzes the formation of acteyl-CoA and oxaloacetate in the cytosol.This reaction is a very important first step for the biosynthesis offatty acids, cholesterol, and acetylcholine, as well as for glucogenesis(Towle et al., 1997). Nutrients and hormones regulate the expressionlevel and phosphorylation status of this key enzyme. Ser454phosphorylation of ACL by Akt and PICA has been reported (Berwick., D CM W et al., 2002; Pierce M W et al., 1982).

The data describes the effect of Coenzyme Q10 on ATP citrate Lyase isthat in normal and cancer cells. It is consistently observed that incancer cells there is a dose-dependent decrease in the expression of ACLenzymes. In contrast there appears to be a trend towards increasedexpression of ACL in normal cells. Cytosolic ACL has been demonstratedto be essential for histone acetylation in cells during growth factorstimulation and during differentiation. The fact that ACL utilizescytosolic glucose derived citrate to generate Acetyl CoA essential forhistone acetylation, a process important in the neoplastic processdemonstrates a role of Coenzyme Q10 induced ACL expression ininfluencing cancer cell function. Acetyl CoA generated from citrate bycytosolic ACL serves as a source for biosynthesis of new lipids andcholesterol during cell division. Thus, Coenzyme Q10 induced changes inACL expression alters Acetyl CoA availability for synthesis of lipidsand cholesterol in normal versus cancer cells. The results aresummarized in the tables below.

TABLE 78 ATPCL in HDFa and MCF-7 Composition Average NormalizedIntensity HDF-Media ~15000 HDF-50-Coenzyme Q10 ~17500 HDF-100-CoenzymeQ10 ~25000 MCF-Media ~7500 MCF-50-Coenzyme Q10 ~7500 MCF-100-CoenzymeQ10 ~12500

TABLE 79 ATP Citrate Lysase ~kd band in HASMC vs. HepG2 Amount -Composition Normalized Intensity HAS5g48-media 24557 HAS5g48-50-CoenzymeQ10 23341 HAS5g48-100-Coenzyme Q10 25544 HAS22G48-Media 27014HAS22G48-50-Coenzyme Q10 21439 HAS22G48-100-Coenzyme Q10 19491Hep5g48-Media 28377 Hep5g48-50-Coenzyme Q10 24106 Hep5g48-100-CoenzymeQ10 22463 Hep22G48-Media 24262 Hep22G48-50-Coenzyme Q10 31235Hep22G48-100-Coenzyme Q10 50588

TABLE 80 ATP Citrate Lysase ~kd band in HASMC vs. PACA2 Amount -Composition Normalized Intensity HAS5g48-media 11036 HAS5g48-50-CoenzymeQ10 12056 HAS5g48-100-Coenzyme Q10 15265 HAS22G48-Media 18270HAS22G48-50-Coenzyme Q10 15857 HAS22G48-100-Coenzyme Q10 13892PACA5g48-Media 11727 PACA5g48-50-Coenzyme Q10 8027 PACA5g48-100-CoenzymeQ10 4942 PACA22G48-Media 8541 PACA22G48-50-Coenzyme Q10 9537PACA22G48-100-Coenzyme Q10 14901

TABLE 81 ATP Citrate Lysase in HepG2 and PACA2 as % of CTR Amount -Composition Normalized Intensity PACA5g48-Media 1.00PACA5g48-50-Coenzyme Q10 0.68 PACA5g48-100-Coenzyme Q10 0.42PACA22G48-Media 1.00 PACA22G48-50-Coenzyme Q10 1.12PACA22G48-100-Coenzyme Q10 1.74 Hep5g48-Media 1.00 Hep5g48-50-CoenzymeQ10 0.85 Hep5g48-100-Coenzyme Q10 0.79 Hep22G48-Media 1.00Hep22G48-50-Coenzyme Q10 1.29 Hep22G48-100-Coenzyme Q10 2.09

Pyruvate Kinase M2 (PKM2)

Pyruvate Kinase is an enzyme involved in the glycolytic pathway. It isresponsible for the transfer of phosphate from phosphoenolpyruvate (PEP)to adenosine diphosphophate (ADP) to generate ATP and pyruvate. PKM2 isan isoenzyme of the glycolytic pyruvate kinase, expression of which ischaracterized by the metabolic function of the tissue i.e. M2 isoenzymeis expressed in normal rapidly proliferating cells with high energyneeds such as embryonic cells and also expressed in few normaldifferentiated tissues such as lung and pancreatic islet cells thatrequire high rate of nucleic acid synthesis. PKM2 is highly expressed intumor cells due to their dependence on glycolytic pathway for meetingcellular energetic requirements. The PKM2 isoform normally thought to beembryonically restricted is re-expressed in cancerous cells. Cellsexpressing PKM2 favor a stronger aerobic glycolytic phenotype (show ashift in metabolic phenotype) with increased lactate production anddecreased oxidative phosphorylation. Thus, decrease in expression ofPKM2 in cancer cells would shift or down-regulate energy generation viathe glycolytic pathway, a strategy that is useful in the treatment ofcancer. Data demonstrates variable expression pattern of PKM2 in normaland cancer cells, with cancer cells demonstrating higher levels ofexpression compared to normal. Treatment of cells with Coenzyme Q10altered expression pattern of the PKM2 upper and lower band levels innormal and cancer cells (FIGS. 81-85). In cancer cells tested, there wasa dose-dependent decrease in the PKM2 expression, and no major changesin normal cells were observed. The results are summarized in the tablesbelow.

TABLE 82 Pyruvate Kinase Muscle form 2 Upper Band in HepG2 NormalizedNormalized Amount - Composition Volume (24 h) Intensity (48 h) 5g-Media28386 413 5g-50-Coenzyme Q10 29269 303 5g-100-Coenzyme Q10 18307 35422G-Media 25903 659 22G-50-Coenzyme Q10 22294 562 22G-100-Coenzyme Q1019560 601

TABLE 83 Pyruvate Kinase Muscle form 2 Lower Band (58 KD) in HepG2Normalized Normalized Amount - Composition Volume (24 h) Volume (48 h)5g-Media 10483 310 5g-50-Coenzyme Q10 11197 185 5g-100-Coenzyme Q10 7642122 22G-Media 9150 306 22G-50-Coenzyme Q10 6302 344 22G-100-Coenzyme Q106904 465

TABLE 84 Pyruvate Kinase Muscle form 2 Upper Band in HASMC Cells afterTreatment Amount - Composition Normalized Intensity 5g48-Media 6085g48-50-Coenzyme Q10 811 5g48-100-Coenzyme Q10 611 22G48-Media 51622G48-50-Coenzyme Q10 595 22G48-100-Coenzyme Q10 496 22G24-Media 30122G24-50-Coenzyme Q10 477 22G24-100-Coenzyme Q10 701

Lactate Dehydrogenase (LDH)

LDH is an enzyme that catalyzes the interconversion of pyruvate andlactate with the simultaneous interconversion of NADH and NAD⁺. It hasthe ability to convert pyruvate to lactate (lactic acid) under low celloxygen tension for generation of reducing equivalents and ATP generationat the expense of mitochondrial oxidative phosphorylation. Cancer cellstypically demonstrate increased expression of LDH to maintain theglycolytic flux to generate ATP and reducing equivalents and reducingmitochondrial OXPHOS. Thus, reducing the expression of the LDH in cancercells would shift metabolism from generation of lactate to facilitateentry of pyruvate into the TCA cycle. Treatment with Coenzyme Q10reduced Lactate Dehydrogenase (LDH) expression in cancer with minimaleffect on normal cells, supporting a role for Coenzyme Q10 in elicitinga shift in cancer cell bioenergtics for the generation of ATP fromglycolytic to mitochondrial OXPHOS sources by minimizing the conversionof cytoplasmic pyruvate to lactic acid. The results are summarized inthe tables below.

TABLE 85 Lactate Dehydrogenase in HepG2 Normalized Normalized Amount -Composition Volume (24 h) Volume (48 h) 5g-Media 7981 59975g-50-Coenzyme Q10 7900 5188 5g-100-Coenzyme Q10 6616 7319 22G-Media9171 7527 22G-50-Coenzyme Q10 7550 6173 22G-100-Coenzyme Q10 7124 9141

TABLE 86 Lactate Dehydrogenase in HepG2 as % Control from 2 ExperimentsAverage Volume as Amount - Composition a % of Control 5g24-Media 1.005g24-50-Coenzyme Q10 0.64 5g24-100-Coenzyme Q10 1.06 5g48-Media 1.005g48-50-Coenzyme Q10 1.12 5g48-100-Coenzyme Q10 1.21 22G24-Media 1.0022G24-50-Coenzyme Q10 1.21 22G24-100-Coenzyme Q10 1.44 22G48-Media 1.0022G48-50-Coenzyme Q10 0.95 22G48-100-Coenzyme Q10 0.67

TABLE 87 Lactate Dehydrogenase in PACA Normalized Normalized Amount -Composition Volume (24 h) Volume (48 h) 5g-Media 2122 23605g-50-Coenzyme Q10 5068 2978 5g-100-Coenzyme Q10 3675 2396 22G-Media4499 2332 22G-50-Coenzyme Q10 10218 2575 22G-100-Coenzyme Q10 7158 3557

Pyruvate Dehydrogenase—B (PDH-E1)

Pyruvate Dehydrogenase beta (PDH-E1) is the first enzyme component thatis part of the pyruvate dehydrogenase complex (PDC) that convertspyruvate to acetyl CoA. PDH-E1 requires thiamine as cofactor for itsactivity, performs the first two biochemical reactions in the PDCcomplex essential for the conversion of pyruvate to acetyl CoA to enterthe TCA cycle in the mitochondria. Thus, concomitant decreases in PKM2and LDH expression along with increase in expression of PDH-E1 in cancercells would enhance the rate of entry of pyruvate towards augmenting themitochondrial OXPHOS for generation of ATP. The data shows that forexpression of PDH-E1 in normal and cancer cell lines, the baselineexpressions of this enzyme is decreased in cancer compared to normalcells. Treatment with Coenzyme Q10 is associated with progressiveincrease in the expression of the PDH-E1 proteins in cancer cells withminimal changes in the normal cells. The results are summarized in thetables below.

TABLE 88 Pyruvate Dehydrogenase Beta in HepG2 Normalized NormalizedAmount - Composition Volume (24 h) Volume (48 h) 5g-Media 517 1005g-50-Coenzyme Q10 921 123 5g-100-Coenzyme Q10 433 205 22G-Media 484 18122G-50-Coenzyme Q10 426 232 22G-100-Coenzyme Q10 340 456

TABLE 89 Pyruvate Dehydrogenase Beta in PACA2 Normalized NormalizedAmount - Composition Volume (24 h) Volume (48 h) 5g-Media 323 3755g-50-Coenzyme Q10 492 339 5g-100-Coenzyme Q10 467 252 22G-Media 572 27622G-50-Coenzyme Q10 924 279 22G-100-Coenzyme Q10 1201 385

TABLE 90 Pyruvate Dehydrogenase Beta in HASMC after Treatmen Amount -Composition Normalized Volume 5g48-Media 140 5g48-50-Coenzyme Q10 1475g48-100-Coenzyme Q10 147 22G48-Media 174 22G48-50-Coenzyme Q10 14922G48-100-Coenzyme Q10 123 22G24-Media 140 22G24-50-Coenzyme Q10 14522G24-100-Coenzyme Q10 150

Caspase 3

Control of the onset of apoptosis is often exerted at the level of theinitiator caspases, caspase-2, -9 and -8/10. In the extrinsic pathway ofapoptosis, caspase-8, once active, directly cleaves and activatesexecutioner caspases (such as caspase-3). The active caspase-3 cleavesand activates other caspases (6, 7, and 9) as well as relevant targetsin the cells (e.g. PARP and DFF). In these studies, the levels ofeffectors caspase-3 protein were measured in the cancer cell lines andin normal cell lines in response to Coenzyme Q10. It should be notedalthough control of apoptosis is through initiator caspases, a number ofsignaling pathways interrupt instead the transmission of the apoptoticsignal through direct inhibition of effectors caspases. For e.g. P38MAPK phosphorylates caspase-3 and suppresses its activity(Alvarado-Kristensson et al., 2004). Interestingly, activation ofprotein phosphates (PP2A) in the same study or protein kinase C delta(PKC delta) (Voss et al., 2005) can counteract the effect of p38 MAPK toamplify the caspase-3 activation and bolster the transmission of theapoptotic signal. Therefore, events at the level of caspase-3 activationor after Caspase 3 activation may determine the ultimate fate of thecell in some cases.

Caspase-3 is a cysteine-aspartic acid protease that plays a central rolein the execution phase of cell apoptosis. The levels of caspase 3 in thecancer cells were increased with Coenzyme Q10 treatment. In contrast theexpression of Caspase-3 in normal cells was moderately decreased innormal cells. The results are summarized in the tables below.

TABLE 91 Caspase 3 in PACA2 Normalized Normalized Amount-CompositionVolume (24 h) Volume (48 h) 5g-Media 324 300 5g-50-Coenzyme Q10 325 7015g-100-Coenzyme Q10 374 291 22G-Media 344 135 22G-50-Coenzyme Q10 675497 22G-100-Coenzyme Q10 842 559

TABLE 92 Caspase 3 in HepG2 cells as % Control from 2 ExperimentsNormalized Volume as Amount - Composition a % of Control 5g24-Media1..00 5g24-50-Coenzyme Q10 1.08 5g24-100-Coenzyme Q10 1.76 5g48-Media1.00 5g48-50-Coenzyme Q10 1.44 5g48-100-Coenzyme Q10 0.95 22G24-Media1.00 22G24-50-Coenzyme Q10 1.39 22G24-100-Coenzyme Q10 1.78 22G48-Media1.00 22G48-50-Coenzyme Q10 1.50 22G48-100-Coenzyme Q10 1.45

TABLE 93 Caspase 3 in HASMC after Treatment Amount - CompositionNormalized Volume 5g48-Media 658 5g48-50-Coenzyme Q10 7665g48-100-Coenzyme Q10 669 22G48-Media 846 22G48-50-Coenzyme Q10 63922G48-100-Coenzyme Q10 624 22G24-Media 982 22G24-50-Coenzyme Q10 83522G24-100-Coenzyme Q10 865

Succinate Dehydrogenase (SDH)

Succinate dehydrogenase, also known as succinate-coenzyme Q reductase isa complex of the inner mitochondrial membrane that is involved in bothTCA and electron transport chain. In the TCA, this complex catalyzes theoxidation of succinate to fumarate with the concomitant reduction ofubiquinone to ubiquinol. (Baysal et al., Science 2000; and Tomlinson etal., Nature Genetics 2002). Germline mutations in SDH B, C and Dsubunits were found to be initiating events of familial paraganglioma orleiomyoma (Baysal et al., Science 2000).

Western blotting analysis was used to characterize expression of SDHSubunit B in mitochondrial preparations of cancer cells treated withCoenzyme Q10. The results suggest that Coenzyme Q10 treatment isassociated with increase SDH protein levels in the mitochondrion of thecells. These results suggest one of the mechanisms of action of CoenzymeQ10 is to shift the metabolism of the cell towards the TCA cycle and themitochondrion by increasing the levels of mitochondrial enzymes such asSDHB. The results are summarized in the table below.

TABLE 94 Succinate Dehydrogenase B in NCIE0808 Mitopreps Composition -Time Average Normalized Volume Media 531 50 uM Coezyme Q10, 3 h 634 100uM Coenzyme Q10, 3 h 964 50 uM Coenzyme Q10, 6 h 1077 100 uM CoenzymeQ10, 6 h 934

Hypoxia Induced Factor-1

Hypoxia inducible factor (Hif) is a transcription factor composed ofalpha and beta subunits. Under normoxia, the protein levels of Hif1alpha are very low owing to its continuous degradation via a sequence ofpost translational events. The shift between glycolytic and oxidativephosphorylation is generally considered to be controlled by the relativeactivities of two enzymes PDH and LDH that determine the catabolic fateof pyruvate. Hif controls this crucial bifurgation point by inducing LDHlevels and inhibiting PDH activity by stimulating PDK. Due to thisability to divert pyruvate metabolism from mitochondrion to cytosol, Hifis considered a crucial mediator of the bioenergetic switch in cancercells.

Treatment with Coenzyme Q10 decreased Hif1 alpha protein levels after inmitochondrial preparations of cancer cells. In whole cell lysates ofnormal cells, the lower band of Hif1a was observed and showed a decreaseas well. The results are summarized in the tables below.

TABLE 95 Hif1 alpha Lower Band in HASMC Cells after Treatment Amount -Composition Normalized Volume 5g48-Media 22244 5g48-50-Coenzyme Q1021664 5g48-100-Coenzyme Q10 19540 22G48-Media 14752 22G48-50-CoenzymeQ10 17496 22G48-100-Coenzyme Q10 23111 22G24-Media 2107322G24-50-Coenzyme Q10 18486 22G24-100-Coenzyme Q10 17919

TABLE 96 Hif1 alpha Upper Band in HepG2 after Treatment Amount -Composition Normalized Volume 5g24-Media 12186 5g24-50-Coenzyme Q10 89985g24-100-Coenzyme Q10 9315 5g48-Media 8868 5g48-50-Coenzyme Q10 86015g48-100-Coenzyme Q10 10192 22G24-Media 11748 22G24-50-Coenzyme Q1014089 22G24-100-Coenzyme Q10 8530 22G48-Media 8695 22G48-50-Coenzyme Q109416 22G48-100-Coenzyme Q10 5608

Example 43 Analysis of Oxygen Consumption Rates (OCR) and ExtracellularAcidification (ECAR) in Normal and Cancer cCells Treated with CoQ10

This example demonstrates that exposure of cells to treatment by arepresentative MIM/epi-shifter of the invention—CoQ10—in the absenceand/or presence of stressors (e.g., hyperglycemia, hypoxia, lacticacid), is associated with a shift towards glycolysis/lactatebiosynthesis and mitochondrial oxidative phosphorylation (as measured byECAR and OCR values) representative of values observed in a normal cellsunder normal physiological conditions.

Applicants have demonstrated in the previous section that treatment withCoQ10 in cancer cells is associated with changes in expression ofspecific proteins that enhance mitochondrial oxidative phosphorylation,with a concomitant decrease in glycolysis and lactate biosynthesis. Thisexample shows that a direct measure of mitochondrial oxidativephosphorylation can be obtained by measuring the oxygen consumptionrates (OCR) in cell lines using the SeaHorse XF analyzer, an instrumentthat measures dissolved oxygen and extracellular pH levels in an invitro experimental model. (SeaHorse Biosciences Inc, North Billerica,Mass.).

The pH of the extracellular microenvironment is relatively acidic intumors compared to the intracellular (cytoplasmic) pH and surroundingnormal tissues. This characteristic of tumors serves multiple purposes,including the ability to invade the extracellular matrix (ECM), ahallmark attribute of tumor metastasis that subsequently initiatessignaling cascades that further modulate:

-   -   tumor angiogenesis    -   increased activation of arrest mechanisms that control cell        cycle turn-over    -   immuno-modulatory mechanisms that facilitate a cellular evasion        system against immunosurveilance    -   metabolic control elements that increase dependency on        glycolytic flux and lactate utilization    -   dysregulation of key apopototic gene families such as Bcl-2,        IAP, EndoG, AIF that serve to increase oncogenicity

While not wishing to be bound by any particular theory, the acidic pH ofthe external microenvironment in the tumor is a consequence of increasein hydrogen ion concentrations extruded from the tumor cells due to theincreased lactate production from an altered glycolytic phenotype.

In this experiment, the OCR and extracellular acidification rate (ECAR)in normal cells lines were obtained in the presence and absence of CoQ10to determine baseline values. It was observed that in its nativenutrient environment, the basal OCR rates in normal cells lines aredifferent, and are usually a function of the physiological roles of thecells in the body.

For example, one set of experiments were conducted using thenon-cancerous cell line HDFa, which is a human adult dermal fibroblastcell line. Fibroblasts are cells that primarily synthesize and secreteextracellular matrix (ECM) components and collagen that form thestructural framework (stroma) for tissues. In addition, fibroblasts areknown to serve as tissue ambassadors of numerous functions such as woundhealing and localized immunomodulation. Under normal physiologicalconditions, energy requirements in normal fibroblasts are met using acombination of glycolysis and oxidative phosphorylation—the glycolysisproviding the necessary nutrients for synthesis of ECM.

In contrast to HDFa, the HASMC (human aortic smooth muscle cell) isfound in arteries, veins, lymphatic vessels, gastrointestinal tracts,respiratory tract, urinary bladder and other tissues with the ability toundergo regulated excitation-contraction coupling. The ability of smoothmuscles such as HASMC cells to undergo contraction requires energyprovided by ATP. These tissues transition from low energy modes whereinATP may be supplied from mitochondria to high energy modes (duringexercise/stress) where energy is provided by switching to glycolysis forrapid generation of ATP. Thus, normal smooth muscle cells can use acombination of mitochondrial OXPHOS and glycolysis to meet their energyrequirements under normal physiological environment.

The differences in their respective physiological roles (i.e., HDFa andHASMC) were observed in the resting OCR values measured in these cellslines using the SeaHorse XF analyzer. FIGS. 37 and 38 below describesthe OCR in HDFa and HASMC cells grown in physiologically normal glucose(about 4.6 mM) and high glucose (hyperglycemic) conditions.

The baseline OCR values for HDFa in the absence of any treatments undernormal oxygen availability is approximately 40 pmoles/min (FIG. 37;above) in the presence of 5.5 mM glucose. This value was slightlyelevated when the cells were maintained at 22 mM glucose. In contrast,in HASMC cells, the OCR values at 5.5 mM glucose is approximately 90pmoles/min, and the OCR value declined to approximately 40 pmoles/minwhile at 22 mM glucose. Thus, under hyperglycemic conditions, there is adifferential response between HDFa and HASMC, further demonstratinginherent differences in their respective physiological make-up andfunction.

Treatment with CoQ10 in cells is associated with changes in OCR that isrepresentative of conditions observed at normal (5 mM) glucoseconditions. The complexity of physiological response is compounded inthe presence of low oxygen tension. Thus, CoQ10 exposure is associatedwith changes in OCR rates in normal cells towards a physiological statethat is native to a particular cell.

Table 97 below describes the ECAR values (mpH/min) in HDFa cells in thepresence or absence of CoQ10 under normoxic and hypoxic conditions at5.5 mM and 22 mM glucose. It can be observed that in normal cells,treatment with CoQ10 had minimal influence on ECAR values, even thoughit influenced OCR in these cells. In high glucose hypoxic conditions,treatment with CoQ10 was associated with lowering of elevated ECAR to avalue that was observed in untreated normoxic conditions.

TABLE 97 ECAR values in HDFa cells in the absence and presence of CoQ10under normoxic and hypoxic conditions at 5.5 mM and 22 mM glucoseNormoxia Hypoxia Normoxia Hypoxia (5.5 mM) (5.5 mM) (22 mM) (22 mM)Treatment ECAR SEM ECAR SEM ECAR SEM ECAR SEM Untreated 5 1.32 5 0.62 50.62 9 0.81 50 μM 6 1.11 5 0.78 5 0.78 6 0.70 31510 100 μM 6 0.76 5 1.195 1.19 8 1.07 31510

In Table 98 the measured baseline ECAR values (mpH/min) in HASMC werehigher compared to that of HDFa. Induction of hypoxic conditions causedan increase in ECAR most likely associated with intracellular hypoxiainduced acidosis secondary to increased glycolysis.

TABLE 98 ECAR values in HASMC cells in the absence and presence of CoQ10under normoxic and hypoxic conditions at 5.5 mM and 22 mM glucoseNormoxic Hypoxic Normoxic Hypoxic (5.5 mM) (5.5 mM) (22 mM) (22 mM)Treatment ECAR SEM ECAR SEM ECAR SEM ECAR SEM Untreated 9 2.22 11 2.1822 2.08 19 1.45 50 μM 9 2.13 11 2.54 21 1.72 17 1.60 31510 100 μM 9 1.7213 2.30 22 1.64 17 1.47 31510

Treatment with CoQ10 was observed to be associated with a downward trendof ECAR rates in hyperglycemic HASMC cells in hypoxic conditions towardsa value that would be observed in normoxic normal glucose conditions.These data demonstrate the presence of physiological variables that isinherent to the physiological role of a specific type of cell,alterations observed in abnormal conditions (e.g. hyperglycemia) isshifted towards normal when treated with CoQ10.

In contrast, cancer cells (e.g., MCF-7, PaCa-2) are inherently primed toculture at higher levels of glucose compared to normal cells due totheir glycolytic phenotype for maintenance in culture. Treatment withCoQ10 caused a consistent reduction in OCR values (FIG. 39 and FIG. 40).

The effects of CoQ10 on OCR values in MCF-7 and PaCa-2 cells was similarto that of the normal HDFa and HASMC cells, wherein the variableresponse was suggestive of a therapeutic response based on individualmetabolic profile of the cancer cell line.

TABLE 99 ECAR values in PaCa-2 cells in the absence and presence ofCoQ10 under normoxic and hypoxic conditions at 5.5 mM and 22 mM glucoseNormoxia Hypoxia Normoxia Hypoxia (17 mM) (17 mM) (22 mM) (22 mM)Treatment ECAR SEM ECAR SEM ECAR SEM ECAR SEM Untreated 21 5.97 16 3.4124 4.35 36 5.65 50 μM 13 3.08 12 1.66 20 5.15 25 4.58 31510 100 μM 142.14 17 2.59 19 3.38 30 5.62 31510

Table 99 describes the ECAR values in PaCa-2 cells. In contrast tonormal cells, cancer cells are phenotypically primed to use high glucosefor ATP generation (enhanced glycolysis) resulting in higher ECAR (Table99, ECAR for untreated normoxia 17 mM) at 21 mpH/min. Treatment withCoQ10 produces a significant decrease in ECAR rates under theseconditions, most likely associated with a decrease in the glycolysisgenerated lactic acid. The associated decrease in OCR in these cells waslikely associated with increased efficiency of the mitochondrial OXPHOS.

A similar comparison of OCR and ECAR values (data not shown) weredetermined in numerous other normal and cancer cells lines, including:HAEC (normal human aortic endothelial cells), MCF-7 (breast cancer),HepG2 (liver cancer) and highly metastatic PC-3 (prostate cancer) celllines. In all of the cell lines tested, exposure to CoQ10 in the absenceand/or presence of stressors (e.g., hyperglycemia, hypoxia, lactic acid)was associated with a shift in OCR and ECAR values representative ofvalues observed in a normal cells under normal physiological conditions.Thus, the overall effect of CoQ10 in the treatment of cancer, includingcell death, is an downstream effect of its collective influence onproteomic, genomic, metabolomic outcomes in concert with shifting of thecellular bioenergetics from glycolysis to mitochondrial OXPHOS.

Example 44 Building Block Molecules for the Biosynthesis of CoQ10

This example demonstrates that certain precursors of CoQ10 biosynthesis,such as those for the biosynthesis of the benzoquinone ring, and thosefor the biosynthesis of the isoprenoid repeats and their attachment tothe benzoquinone ring (“building block components”), can be individuallyadministered or administered in combination to target cells, and effectdown-regulation of the apoptosis inhibitor Bcl-2, and/or up-regulationof the apoptosis promoter Caspase-3. Certain precursors or combinationsthereof may also inhibit cell proliferation. The data suggests that suchCoQ10 precursors may be used in place of CoQ10 to achieve substantiallythe same results as CoQ10 administration.

Certain exemplary experimental conditions used in the experiments arelisted below.

Skmel-28 melanoma cells were cultured in DMEM/F12 supplemented with 5%Fetal Bovine Serum (FBS) and 1X final concentration of Antibiotics. Thecells were grown to 85% confluency and treated with building blockcomponents for 3, 6, 12 and 24 hours. The cells were then pelleted and aWestern blot analysis was performed. The test building block componentsincluded L-Phenylylalanine, DL-Phenylyalanine, D-Phenylylalanine,L-Tyrosine, DL-Tyrosine, D-Tyrosine, 4-Hydroxy-phenylpyruvate,phenylacetate, 3-methoxy-4-hydroxymandelate (vanillylmandelate or VMA),vanillic acid, 4-hydroxy-benzoate, pyridoxine, panthenol, mevalonicacid, Acetylglycine, Acetyl-CoA, Farnesyl, and2,3-Dimethoxy-5-methyl-p-benzoquinone.

In the Western Blot Analysis, the cells were pelleted in cold PBS,lysed, and the protein levels were quantified using a BCA protein assay.The whole cell lysate was loaded in a 4% loading 12% running Tris-HClgel. The proteins were then transferred to a nitrocellulose paper thenblocked with a 5% milk Tris-buffered solution for 1 hour. The proteinswere then exposed to primary antibodies (Bcl-2 and Caspase-3) overnight.The nitrocellulose paper was then exposed to Pico Chemilluminescent for5 min and the protein expression was recorded. After exposure, actin wasquantified using the same method. Using ImageJ the levels of proteinexpression were quantified. A t-Test was used to analyze for statisticalsignificance.

Illustrative results of the experiments are summarized below.

Western Blot Analysis of Building Block component L-Phenylalanine:Before proceeding to the synthesis pathway for the quinone ringstructure, L-Phenylalanine is converted to tyrosine. A western blotanalysis was performed to quantify any changes in the expression of theapoptotic proteins in the melanoma cells. The concentrations tested were5 μM, 25 μM, and 100 μM. Initial studies added L-Phenylalanine toDMEM/F12 medium which contained a concentration of 0.4 M phenylalanine.For the 5 μM, 25 μM, and 100 μM the final concentration of theL-Phenylalanine in the medium was 0.405 M, 0.425 M, and 0.500 M,respectively. These final concentrations were tested on the Skmel-28cells for incubation periods of 3, 6, 12 and 24 hours. The cells weregrown to 80% confluency before adding the treatment medium and harvestedusing the western blot analysis procedure as described above. Astatistically significant decrease in Bcl-2 was observed for the 100 μML-Phenylalanine after 3 hours and 12 hours incubation. Fr the 5 μML-phenylalanine, a statistically significant decrease in Bcl-2 wasobserved after 6 hours of incubation. For the 25 μM L-phenylalanine, astatistically significant decrease in Bcl-2 and a statisticallysignificant increase in Caspase-3 were observed after 12 hours ofincubation. A statistically significant decrease in Bcl-2 indicates achange in the apoptotic potential and a statistically significantincrease in Caspase-3 confirms the cells are undergoing apoptosis. Therewas a constant trend for the decrease in Bcl-2 compared to the controleven though, due to sample size and standard deviation, these timepoints were not statistically significant in this experiment.

Western Blot Analysis of Building Block component D-Phenylalanine:D-Phenylalanine, a chemically synthetic form of the bioactiveL-Phenylalanine, was tested for comparison to L-phenylalanine. For allthree concentrations (5 μM, 25 μM, and 100 μM of D-Phenylalanine, therewas a significant reduction in Bcl-2 expression after 6 hours ofincubation. In addition, for the 5 μM and 25 μM, there was a significantreduction after 3 hours of incubation. For the 5 μM and 100 μMconcentrations, a significant increase in Caspase-3 expression wasobserved after 6 hours of incubation.

Western Blot Analysis of Building Block component DL-Phenylalanine:DL-Phenylalanine was also tested for comparison to L-Phenylalanine.Again, concentrations of 5 μM, 25 μM, and 100 μM were tested on Skmel-28cells. The incubation periods were 3, 6, 12 and 24 hours. Astatistically significant increase in Caspase-3 was observed after 3hours of incubation. A statistically significant decrease in Bcl-2 wasobserved after 24 hours of incubation. Although a decreasing Bcl-2 andincreasing Caspase-3 trend at all other concentrations and incubationtime points, they were not statistically significant in this experiment.

Western Blot Analysis of Building Block component L-Tyrosine: L-Tyrosineis a building block component for the synthesis of quinone ringstructure of CoQ10. Initial testing of L-Tyrosine did not result in ahigh enough protein concentration for western blot analysis. From thisstudy concentrations under 25 μM were tested for Western Blot Analysis.The DMEM/F12 medium used contained L-Tyrosine disodium saltconcentration of 0.398467 M. The initial concentration was increased by500 nM, 5 μM, and 15 μM. A statistically significant increase inCaspase-3 was observed for the 500 nM concentration after 12 hours ofincubation. A statistically significant increase in Caspase-3 was alsoobserved for the 5A statistically significant decrease in Bcl-2 wasobserved for the 5 μM concentration after 24 hours of incubation. Astatistically significant decrease in Bcl-2 was observed for the 500 μMand 5 μM concentrations after 24 hours of incubation.

Western Blot Analysis of Building Block component D-Tyrosine:D-Tyrosine, a synthetic form of L-Tyrosine, was tested for comparisonagainst the L-Tyrosine apoptotic effect on the melanonal cells. Based oninitial studies with L-Tyrosine, concentrations below 25 μM were chosenfor the western blot analysis. The concentrations tested were 1 μm, 5μM, and 15 μM. D-Tyrosine showed a reduction in Bcl-2 expression for the5 μM and 15 μM concentrations for 12 and 24 hour time periods. Caspase-3was significantly increased for the concentration of 5 μM for 3, 12 and24 time periods. Also there was an increase in Caspase-3 expression forthe 1 μM for 12 and 24 hour time period. In addition there is anincrease in Caspase-3 expression for 5 μM for the 12 hour time period.

Western Blot Analysis of Building Block component DL-Tyrosine:DL-Tyrosine, a synthetic form of L-Tyrosine, was also tested forcomparison against L-Tyrosine's apoptotic effect on the cells. There isa statistical decrease in Bcl-2 expression seen in the 1 μM and 15 μMconcentrations after 12 hours incubation and for the 5 μM after 24 hourof incubation. An increase in Caspase-3 expression was also observed forthe 5 μM and 15 μM after 12 hours of incubation.

Western Blot Analysis of Building Block component4-Hydroxy-phenylpyruvate: 4-Hydroxy-phenylpyruvate is derived fromTyrosine and Phenylalanine amino acids and may play a role in thesynthesis of the ring structure. The concentration of 1 μM, 5 μM, and 15μM were tested for Bcl-2 and Caspase-3 expression. For the 5 μM and 15μM concentrations there is a significant reduction in Bcl-2 expressionafter 24 hours of incubation and a significant increase in Caspase-3expression after 12 hours of incubation.

Western Blot Analysis of Building Block component Phenylacetate:Phenylacetate has the potential to be converted to 4-Hydroxy-benzoate,which plays a role in the attachment of the side chain to the ringstructure. The concentration tested were 1 μM, 5 μM, and 15 μM. Forphenylacetate there was a decrease in Bcl-2 expression for theconcentration of 5 μM and 15 μM after 12 hours and 24 hours ofincubation. An increase in Caspase-3 expression was observed for theconcentration of 5 μM and 15 μM after 12 hours and 24 hours ofincubation.

Western Blot Analysis of Building Block component3-methoxy-4-hydroxymandelate (vanillylmandelate or VMA): VMA is anadditional component for the synthesis of the CoQ10 quinone ringstructure. The concentrations tested were 100 nM, 250 nM, 500 nM, 1 μM,25 μM, 50 μM, and 100 μM. Though no statistically significant apoptoticeffect was observed in this experiment, the data indicated a downwardtrend of Bcl-2 expression.

Western Blot Analysis of Building Block component Vanillic acid:Vanillic is a precursor for the synthesis of the quinone ring and wastested at a concentration of 500 nm, 5 μM, and 15 μM. A western blotanalysis measured Bcl-2 and Caspase-3 expression. Vanillic Acid wasshown to significantly reduce Bcl-2 expression for the concentrations of500 nM and 5 μM at the 24 hour incubation time point. For the 15 μMconcentration there is a reduction in Bcl-2 expression after 3 hours ofincubation. For the cells incubated with 15 μM for 24 hours there was asignificant increase in Caspase-3 expression.

Western Blot Analysis of Building Block component 4-Hydroxybenzoate:4-Hydroxybenzoate acid plays a role in the attachment of the isoprenoidside chain to the ring structure. The concentrations tested were 500 nM,1 μM, and 50 μM. There was a significant reduction in Bcl-2 expressionfor the 15 μM concentration after 24 hours of incubation.

Western Blot Analysis of Building Block component 4-Pyridoxine:Pyridoxine is another precursor building block for the synthesis of thequinone ring structure of CoQ10. The concentrations tested for thiscompound are 5 μM, 25 μM, and 100 μM. The cells were assayed for theirlevels of Bcl-2 and Caspase-3. Pyridoxine showed a significant reductionin Bcl-2 after 24 hours of incubation in melanoma cells.

Western Blot Analysis of Building Block component Panthenol: Panthenolplays a role in the synthesis of the quinone ring structure of CoQ10.The concentrations tested on melanoma cells were 5 μM, 25 μM, and 100μM. This compound showed a significant reduction in Bcl-2 expression forthe 25 μM concentration.

Western Blot Analysis of Building Block component Mevalonic: MevalonicAcid is one of the main components for the synthesis of CoQ10. Thiscompound was tested at the concentrations of 500 nM, 1 μM, 25 μm, and 50μM. There was no significant reduction in Bcl-2 expression or anincrease in Caspase-3 expression in this experiment.

Western Blot Analysis of Building Block component Acetylglycine: Anotherroute for the synthesis of CoQ10 is the isoprenoid (side chain)synthesis. The addition of Acetylglycine converts Coenzyme A toAcetyl-CoA which enters the mevalonic pathway for the synthesis of theisoprenoid synthesis. The concentrations tested were 5 μM, 25 μM, and100 μM. The testing of Acetylglycine showed significant decrease inBcl-2 expression after 12 hours of incubation for the concentration of 5μM and 25 μM. A significant decrease in Bcl-2 was recorded for the 100μM concentration at the 24 hour incubation time point.

Western Blot Analysis of Building Block component Acetyl-CoA: Acetyl-CoAis a precursor for the mevalonic pathway for the synthesis of CoQ10. Theconcentrations tested were 500 nm, 1 μM, 25 μM, and 50 μM. There was nosignificant observed reduction in Bcl-2 or increase in Caspase-3expression for the time points and concentrations tested.

Western Blot Analysis of Building Block component L-Tyrosine incombination with farnesyl: L-Tyrosine is one of the precursors for thesynthesis of the quinone ring structure for CoQ10. Previous experimenttested the reaction of L-Tyrosine in medium with L-Phenylalanine andL-Tyrosine. In this study L-Tyrosine was examined in medium without theaddition of L-Phenylalanine and L-Tyrosine. In this study the finalconcentrations of L-Tyrosine tested were 500 nM, 5 μM, and 15 μM.Farnesyl was tested at a concentration of 50 μM. There was no observedsignificant response for the 3 and 6 hour time points.

Western Blot Analysis of Building Block component L-Phenylalanine incombination with Farnesyl: L-Phenylalanine, a precursor for thesynthesis of the quinone ring structure, was examine in combination withfarnesyl in medium free of L-Tyrosine and L-Phenylalanine. A westernblot analysis was performed to assay the expression of Bcl-2 andCaspase-3. The final concentrations of L-Phenylalanine were: 5 μM, 25μM, and 100 μM. Farnesyl was added at a concentration of 50 μM. Thisstudy showed a decrease in Bcl-2 expression for most of theconcentrations and combinations tested as depicted in the table below.

L- 3 hr 6 hr 12 hr 24 hr Phenylalanine Bcl-2 Cas-3 Bcl-2 Cas-3 Bcl-2Cas-3 Bcl-2 Cas-3 5 μM X 5 μM w/ X X Farnesyl 25 μM X X 25 μM w/ X XFarnesyl 100 μM X X X 100 μM w/ X Farnesyl

Cell Proliferation Assay of the Combination of 4-Hydroxy-Benzoate withBenzoquinone: This set of experiments used a cell proliferation assay toassess the effect of combining different building block molecules oncell proliferation.

The first study examined the effect of combining 4-Hydroxy-Benzoate withBenzoquinone. Cells were incubated for 48 hours, after which a cellcount was performed for the live cells. Each test group was compared tothe control, and each combination groups were compared to Benzoquinonecontrol. The compounds were statistically analyzed for the addition ofBenzoquinone. The following table summarizes the cell count resultswherein the X mark indicates a statistical decrease in cell number.

Compared to 4- Hydroxy to Compared to Compared compound w/o Benzoquinone4-Hydroxy to Ctrl Benzoquinone Control 500 nm X 500 nm w/Benzo X X (35μM) 500 nm w/Benzo X X (70 μM) 1 μm X 1 μm w/Benzo X X (35 μM) 1 μmw/Benzo X X (70 μM) 50 μm X 50 μm w/Benzo X (35 μM) 50 μm w/Benzo X X X(70 μM)

There is a significant decrease in cell number for the cells incubatedwith 4-Hydroxybenzoic and benzoquinone and in combination. For thecombination of 50 μM 4-Hydroxybenzoate in combination with 70 μMBenzoquinone there is significant reduction in cell number compared tothe Benzoquinone control. This suggests a synergistic effect for thismolar ratio.

Additional studies were performed testing additional molar ratios. Forthe first test 4-Hydroxybenzoic were tested at concentrations of 500 nM,1 μM, and 50 μM. These concentrations were tested in combination with2,3-Dimethoxy-5-methyl-p-benzoquinone (Benzo). The concentration ofBenzo tested were 25 μM, 50 μM, and 100 μM. Melanoma cells were grown to80% confluency and seeded in 6 well plates at a concentration of 40Kcells per well. The cells were treated with CoQ10, 4-Hydroxybenzoate,Benzo, and a combination of 4-Hydroxybenzoate/Benzo.

A T-test was performed with p<0.05 as statistically significant. An Xsignifies a statistical decrease in cell number.

Ctrl vs Benzo 25 μM X Ctrl vs Benzo (B) 50 μM Ctrl vs Benzo (B) 100 uM XCtrl vs 4-Hydroxybenzoate (HB) 500 nm X Ctrl vs HB 1 μM X Ctrl vs HB 50μM X 500 nM HB vs 500 nM HB w/25 B X 500 nM HB vs 500 nM HB w/50 B X 500nM HB vs 500 nM HB w/100 B X 1 uM HB vs 1 μM HB w/25 B X 1 uM HB vs 1 μMHB w/50 B X 1 uM HB vs 1 μM HB w/100 B 50 uM HB vs 50 μM HB w/25 B X 50uM HB vs 50 μM HB w/50 B X 50 uM HB vs 50 μM HB w/100 B 500 nM HB w/25 Bvs 25 B X 500 nM HB w/50 B vs 50 B X 500 nM HB w/100 B vs 100 B X 1 μMHB w/25 B vs 25 B X 1 μM HB w/50 B vs 50 B X 1 μM HB w/100 B vs 100 B 50μM HB w/25 B vs 25 B X 50 μM HB w/50 B vs 50 B X 50 μM HB w/100 B vs 100B

There is a significant decrease in cell proliferation for the treatmentmedium containing HB. Moreover the combination of the HB withbenzoquinone showed a significant reduction in cell number compare tothe cells incubated with the corresponding benzoquinone concentrations.

A cell proliferation assay was also performed on neonatal fibroblastcells. The concentrations of HB tested were 500 nM, 5 μM, and 25 μM. HBwas also tested in combination with benzoquinone at a concentrations of25 μM, 50 μM, and 100 μM. Melanoma cells were seeded at 40 k cells perwell and were treated for 24 hours. The cells were trypsinized andquantified using a coulter counter.

Statistical analysis did not show a significant reduction in fibroblastcells. This indicates minimal to no toxicity in normal cells.

Cell Proliferation Assay of the Combination of phenylacetate andbenzoquinone: Phenyl acetate is a precursor for the synthesis of4-Hydroxybenzoic acid (facilitates the attachment of the ring structure.A cell proliferation assay was performed to assay the effect ofincubating phenylacetate in combination with CoQ10 and Benzoquinone.

Ctrl and 25/25 μM Ben X Ctrl and 25/50 μM Ben X Ctrl and 25/100 μM Ben XCtrl and 25/25 μM Q-10 X Ctrl and 25/25 μM Q-10 X Ctrl and 25/50 μM Q-10X Ctrl and 25/100 μM Q-10 X Ctrl and Ben 25 X Ctrl and Ben 50 X Ctrl andBen 100 X Ctrl and Q-10 25 Ctrl and Q-10 50 Ctrl and Q-10 100 X Ben 25μM and 500 nM/25 μM Ben X Ben 25 μM and 5 nM/25 μM Ben X Ben 25 μM and25 nM/25 μM Ben X Ben 50 μM and 500 nM/50 μM Ben X Ben 50 μM and 5 nM/50μM Ben X Ben 50 μM and 25 nM/50 μM Ben X Ben 100 μM and 500 nM/100 μMBen Ben 100 μM and 5 nM/100 μM Ben Ben 100 μM and 25 nM/100 μM Ben Q-1025 μM and 500 nM/25 μM Q-10 X Q-10 25 μM and 5 nM/25 μM Q-10 X Q-10 25μM and 25 nM/25 μM Q-10 X Q-10 50 μM and 500 nM/50 μM Q-10 X Q-10 50 μMand 5 nM/50 μM Q-10 X Q-10 50 μM and 25 nM/50 μM Q-10 X Q-10 100 μM and500 nM/100 μM Q-10 X Q-10 100 μM and 5 nM/100 μM Q-10 X Q-10 100 μM and25 nM/100 μM Q-10 X

The data indicates the addition of phenylacetate in combination withbenzoquinone significantly decreases the cellular proliferation. Thecombination with CoQ10 and phenylacetate significantly decrease the cellnumber compared to incubation with CoQ10 and benzoquinone alone.

Cell Proliferation Assay of the Combination of 4-Hydroxy-Benzoate withFarnesyl: 4-Hydroxy-Benzoate was incubated in combination with Farnesyl.The summary of the results are listed below. 4-Hydroxybenzoate groupswere compared to the control and Farnesyl control groups. The Xsignifies a statistical decrease in cell number.

Compared to 4- Hydroxy to 4-Hydroxy- Compared compound w/o Compared toBenzoate to Ctrl Farnesyl Farnesyl Control 500 nm X 500 nm w/Farnesyl X(35 μM) 500 nm w/Farnesyl X (70 μM) 1 μm Error 1 μm w/Farnesyl Error (35μM) 1 μm w/Farnesyl Error (70 μM) 50 μm X 50 μm w/Farnesyl X (35 μM) 50μm w/Farnesyl X (70 μM)

Cell Proliferation Assay of the Combination of L-Phenylalanine withBenzoquinone: A cell proliferation assay was performed to test thecombination of L-Phenylalanine combined with Benzoquinone. Below is asummary of the results of L-Phenylalanine compared to the control andBenzoquinone control. The X signifies a statistical decrease.

Compared to L- Phenylalanine to Compared to Compared compound w/oBenzoquinone L-Phenylalanine to Ctrl Benzoquinone Control 5 μM 5 μmw/Benzo X (50 μM) 5 μm w/Benzo X (100 μM) 25 μm 25 μm w/Benzo X (50 μM)25 μm w/Benzo X (100 μM) 100 μm 100 μm w/Benzo X X X (50 μM) 100 μmw/Benzo X X X (100 μM)

A similar synergistic role is seen for the L-Phenylalanine combined withBenzoquinone.

Cell Proliferation Assay of the Combination of L-Phenylalanine withFarnesyl: Preliminary results for combination cell proliferation studyof L-Phenylalanine incubated in combination with Farnesyl. TheL-Phenylalanine were compared to the control and Farnesyl control group.An X signifies a statistical decrease in cell number.

Compared to L- Phenylalanine to Compared compound w/o Compared toL-Phenylalanine to Ctrl Farnesyl Farnesyl Control 5 μM 5 μm w/Farnesyl(50 μM) 5 μm w/Farnesyl (100 μM) 25 μm X 25 μm w/Farnesyl X X X (50 μM)25 μm w/Farnesyl X X X (100 μM) 100 μm X 100 μm w/Farnesyl X X (50 μM)100 μm w/Farnesyl X (100 μM)

Cell Proliferation Assay of the Combination of L-Tyrosine withBenzoquinone: L-Tyrosine was incubated in combination with Benzoquinoneafter which a cell count was performed. The groups were compared thecontrol groups and Benzoquinone control group.

Compared to L- Tyrosine to Compared to Compared compound w/oBenzoquinone L-Tyrosine to Ctrl Benzoquinone Control 500 nm 500 nmw/Benzo (50 μM) 500 nm w/Benzo (100 μM) 5 μm X 5 μm w/Benzo X (50 μM) 5μm w/Benzo X (100 μM) 15 μm X 15 μm w/Benzo X (50 μM) 15 μm w/Benzo X(100 μM)

The addition of Benzoquinone did not amplify the effect of L-Tyrosine onthe cell number.

Cell Proliferation Assay of the Combination of L-Tyrosine withBenzoquinone: This study examined the combination of L-Tyrosine withFarnesyl. The groups were compared to control and Farnesyl controlgroups.

Compared to L- Tyrosine to Compared compound w/o Compared to L-Tyrosineto Ctrl Farnesyl Farnesyl Control 500 nm 500 nm w/Farnesyl (50 μM) 500nm w/Farnesyl (50 μM) 5 μm X 5 μm w/Farnesyl X (50 μM) 5 μm w/Farnesyl X(100 μM) 15 μm X 15 μm w/Farnesyl X (50 μM) 15 μm w/Farnesyl X (100 μM)

Combining L-Tyrosine and Farnesyl does not appear to have a synergisticeffect on reducing the cell number in this experiment.

The synthesis of the CoQ10 is divided into two main parts, which consistof the synthesis of the ring structure and synthesis of the side chainstructure. Here, oncogenic cells were supplemented with compounds whichare precursors for the synthesis of the side chain and the ringstructure components. Our results have focused the study to 3 maincomponents involved in the synthesis of the ring structure and twocompounds that play a role in the attachment of the ring structure tothe side chain structure. The three compounds that have shown asignificant reduction in Bcl-2 and increase in Caspase-3 expressionare: 1) L-Phenylalanine, 2) L-Tyrosine and 3) 4-Hydroxyphenylpyruvate.The two compounds involved with the attachment of the side chain to thering structure are: 1) 4-hydroxy benzoate and 2) Phenylacetate.

Our results also showed that exogenous delivery of these compounds incombination with 2,3 Dimethoxy-5-methyl-p-benzoquinone (benzoquinone)significantly inhibits cell proliferation. This indicates asupplementation of the ring structure with compounds for the attachmentof the side chain to the benzoquinone ring may supplement an impairedCoQ10 synthesis mechanism. This may also assist in the stabilization ofthe molecule to maintain the functional properties required by cellularprocesses. Phenylacetate is a precursor for the synthesis of4-Hydroxybenzoate, which exogenous delivery in combination withbenzoquinone has a similar effect in oncogenic cells.

Example 45 Modulation of Gene Expression by Coenzyme Q10 in Cell Modelfor Diabetes

Coenzyme Q10 is an endogenous molecule with an established role in themaintenance of normal mitochondrial function by directly influencingoxidative phosphorylation. Experimental evidence is presented thatdemonstrates the ability of Coenzyme Q10 in modulating intracellulartargets that serve as key indices of metabolic disorders, such asdiabetes, in a manner representative of therapeutic endpoints.

In order to understand how Coenzyme Q10 regulates expression of genesassociated with the cause or treatment of diabetes, immortalized primarykidney proximal tubular cell line derived from human kidney (HK-2) andprimary cultures of the human aortic smooth muscle cells (HASMC) wereused as experimental models. The HK-2 and HASMC cells are normallymaintained in culture at 5.5 mM glucose, which is a concentration thatcorresponds to a range considered normal in human blood. However, inorder to simulate a diabetic environment, both cell lines weresubsequently maintained at 22 mM glucose, which corresponds to the rangeobserved in human blood associated with chronic hyperglycemia. The cellswere subsequently allowed to propagate over 3 passages so that theintracellular regulation processes were functionally adapted to mimic adiabetic state. The choice of cell line was based on the physiologicinfluence of diabetes on renal dysfunction and progression to end-stagerenal disease (ESRD) in addition to the progressive pathophysiology of acompromised cardiovascular function.

Effect of Coenzyme Q10 on Gene Expression in HK-2 Cells Using theDiabetes PCR Array

The Diabetes PCR array (SABiosciences) offers a screen for 84 genessimultaneously. The 4 treatments tested in this study were:

-   -   HK-2;    -   HK-2 H maintained 22 mM glucose;    -   HK2(H)+50 μM Coenzyme Q10; and    -   HK2(H)+100 μM Coenzyme Q10.

A stringent analysis of the Real time PCR data of the HK-2 samples onthe Diabetes Arrays (Cat #PAHS-023E, SABiosciences Frederick Md.) wasmade to exclude all results where gene regulation was not at least atwo-fold regulation over HK-2 normal untreated cells with a p value ofless than 0.05. Genes that were observed to be regulated either bychronic hyperglycemia or by Coenzyme Q10 are listed in Table 100 andtheir functions and subcellular locations (derived from IngenuityPathway Analysis) are listed in Table 101.

TABLE 100 HK-2(H)-50 μM HK-2(H)-100 μM HK-2(H) p Coenzyme Q10 p CoenzymeQ10 p Genes Fold regulation value Fold regulation value Fold regulationvalue CEACAM1 1.26 0.409 3.47 0.067 5.36 0.032 PIK3C2B 1.48 0.131 2.320.115 3.31 0.003 INSR −1.09 0.568 2.51 0.103 2.88 0.024 TNF 2.00 0.0052.57 0.042 2.81 0.020 ENPP1 −1.50 0.002 1.42 0.238 2.67 0.038 PRKCB−1.75 0.005 1.82 0.280 2.49 0.042 DUSP4 1.27 0.318 1.24 0.455 2.26 0.060SELL −1.58 0.219 1.77 0.042 2.06 0.021 SNAP25 −1.00 0.934 1.46 0.3771.97 0.059

TABLE 101 Symbol Entrez Gene Name Location Type(s) CEACAM1carcinoembryonic antigen- Plasma trans- related cell adhesion mole-Membrane membrane cule 1 (biliary glycoprotein) receptor PIK3C2Bphosphoinositide-3-kinase, Cytoplasm kinase class 2, beta polypeptideINSR insulin receptor Plasma kinase Membrane TNF tumor necrosis factor(TNF Extracellu- cytokine superfamily, member 2) lar Space ENPP1ectonucleotide pyrophosphatase/ Plasma enzyme phosphodiesterase 1Membrane PRKCB protein kinase C, beta Cytoplasm kinase DUSP4 dualspecificity phosphatase 4 Nucleus phos- phatase SELL selectin L Plasmaother Membrane SNAP25 synaptosomal-associated protein, Plasma trans- 25kDa Membrane porter

Among the detected RNA transcripts with modulated levels, the CarcinoEmbryonic Antigen Cell Adhesion Molecule 1 (CEACAM1) was identified asbeing highly upregulated in HK2(H) cells, particularly with 100 μMCoenzyme Q10 treatment. CEACAM-1, also known as CD66a and BGP-I, is a115-200 KD type 1 transmembrane glycoprotein that belongs to themembrane-bound CEA subfamily of the CEA superfamily. On the surface ofcells, it forms noncovalent homo- and heterodimers. The extracellularregion contains three C2-type Ig-like domains and one N-terminal V-typeIg-like domain. Multiple splice variants involving regions C-terminal tothe second C2-type domain (aa 320 and beyond) exist. The lack of intactCEACAM1 expression in mice has been proposed to promote the metabolicsyndrome associated with diabetes, while an increase in expression ofCEACAM1 is associated with increased insulin internalization, whichsuggests an increase in insulin sensitivity and glucose utilization(e.g., movement of glucose from blood into the cells), thus mitigatinginsulin resistance, a hallmark characteristic of type 2 diabetesmellitus.

As shown in Table 100, insulin receptor (INSR) expression was alsoaltered in diabetic HK-2 cells treated with Coenzyme Q10. Without beingbound by theory, the increase in expression of INSR with Coenzyme Q10treatment should enhance insulin sensitivity (either alone or inaddition to expression of CEACAM1) with the potential to reverse a majorphysiologic/metabolic complication associated with diabetes.

Effect of Coenzyme Q10 on Gene Expression in HK-2 Cells UsingMitochondrial Arrays

Differential expression of mitochondrial genes in diabetes was assayedusing the mitochondria arrays (Cat#PAHS 087E, SABisociences FrederickMd.). Genes that were regulated by chronic hyperglycemia and/or CoenzymeQ10 treatment are listed in Table 102 while their functions and locationare included in Table 103.

TABLE 102 HK2 (H) p HK-2(H) 50 μM p HK-2(H) 100 μM p Genes untreatedvalue Coenzyme Q10 value Coenzyme Q10 value GRPEL1 −1.5837 0.151255−2.6512 0.04704 −1.933 0.139161 SLC25A3 −8.6338 0.071951 −8.2059 0.0425−1.6984 0.995194 TOMM40 −2.3134 0.140033 −1.1567 0.115407 −1.95090.038762 TSPO −3.6385 0.111056 −6.7583 0.073769 −2.1104 0.167084

TABLE 103 Symbol Entrez Gene Name Location Type(s) GRPEL1 GrpE-like 1,mitochon- Mitochondria other drial (E. coli) SLC25A3 solute carrierfamily Mitochondrial transporter 25 (mitochondrial membrane. carrier;phosphate carrier), member 3 TOMM40 translocase of outer Outer membraneion channel mitochondrial membrane of mitochondria. 40 homolog (yeast)TSPO translocator protein Outer membrane transmembrane (18 kDa) ofmitochondria. receptor

To date, the role of the four mitochondrial genes identified (Table 102)in diabetic HK-2 cells treated with Coenzyme Q10 in diabetes isuncharacterized.

Study 2: Effect of Coenzyme 010 on Gene Expression in HASMC Cells Usingthe Diabetes PCR Array

The Diabetes PCR array (SABiosciences) offers a screen for 84 genessimultaneously. The 4 treatments tested in this study were:

-   -   HASMC;    -   HASMC H maintained at 22 mM glucose;    -   HASMC(H)+50 μM Coenzyme Q10; and    -   HASMC(H)+100 μM Coenzyme Q10.

A stringent analysis of the Real time PCR data of the HASMC cell sampleson the Diabetes Arrays (Cat #PAHS-023E, SABiosciences Frederick Md.) wasmade to exclude all results where gene regulation was not at least atwo-fold regulation over HASMC normal untreated cells with a p value ofless than 0.05. Genes that were observed to be regulated either bychronic hyperglycemia or by Coenzyme Q10 are listed in Table 104.

TABLE 104 p HASMC-(H)-50 μM p HASMC-(H)-100 μM p Genes HASMC-(H) valueCoenzyme Q10 value Coenzyme Q10 value AGT 1.3051 0.547507 −1.01690.781622 2.3027 0.030195 CCL5 −17.4179 0.013798 −5.3796 0.022489 −4.69130.022696 CEACAM1 −5.5629 0.012985 −5.3424 0.014436 −5.8025 0.012948 IL62.7085 0.049263 3.8172 0.012685 6.0349 0.000775 INSR 1.4649 0.2077881.9622 0.081204 2.0801 0.016316 NFKB1 1.482 0.072924 1.3779 0.1911912.0898 0.027694 PIK3C2B 2.0479 0.218276 1.4331 0.254894 2.6329 0.069422SELL −1.9308 0.087513 1.2476 0.393904 4.0371 0.000177 TNF −1.8140.108322 −3.2434 0.043526 −1.8489 0.133757

In HASMC cells, treatment of hyperglycemic cells with Coenzyme Q10resulted in the altered expression of genes involved in regulatingvascular function (AGT), insulin sensitivity (CEACAM1, INSR, SELL) andinflammation/immune function (IL-6, TNF, CCL5). Without being bound bytheory, an increase in expression of INSR may be associated withincreased insulin sensitivity in HASMC cells, which is a physiologicalproperty that would be beneficial in the treatment of diabetes, whileIL-6, in addition to its immunoregulatory properties, has been proposedto affect glucose homeostasis and metabolism, both directly andindirectly, by action on skeletal muscle cells, adipocytes, hepatocytes,pancreatic β-cells and neuroendocrine cells. Upon activation, normalT-cell express and secrete RANTES and chemokine (C-Cmotif) ligand(CCL5). CCL5 is expressed by adipocytes, and serum levels of RANTES areincreased in obesity and type 2 diabetes. However, as shown in Table104, treatment of HASMC cells with Coenzyme Q10 causes a significantdecrease in the expression of CCL5. Based on the foregoing data, it isexpected that administration of Coenzyme Q10 will have a therapeuticbenefit in the management of diabetes.

Effect of Coenzyme Q10 on Gene Expression in HASMC Cells UsingMitochondrial Arrays

Differential expression of mitochondrial genes in diabetes was assayedusing the mitochondria arrays (Cat#PAHS 087E, SABisociences FrederickMd.). Genes that were regulated by chronic hyperglycemia and/or CoenzymeQ10 treatment are shown in Table 105.

TABLE 105 p HASMC-(H) 50 uM p HASMC-(H)-100 μM p Genes HASMC-(H) valueCoenzyme Q10 value Coenzyme Q10 value BCL2L1 −1.6558 0.244494 −2.78630.008744 −2.3001 0.014537 MFN1 −1.4992 0.317009 −1.2585 0.021185 −2.26320.005961 PMAIP1 −4.7816 0.206848 −6.8132 0.000158 −4.352 0.000286SLC25A1 −2.2051 0.020868 −1.834 0.00581 −3.0001 0.03285 SLC25A13 −2.05270.035987 −1.5 0.029019 −1.5245 0.043712 SLC25A19 −1.0699 0.417217−1.4257 0.104814 −2.1214 0.007737 SLC25A22 −2.1747 0.007344 −1.98390.0013 −10.3747 0.003437 TIMM44 −1.3605 0.414909 −2.3214 0.004118−1.9931 0.010206 TOMM40 −1.1982 0.428061 −2.0922 0.002195 −2.26840.003272 TSPO −1.402 0.304875 −2.0586 0.061365 −2.3647 0.044656

Treatment of hyperglycemic HASMC cells with Coenzyme Q10 resulted inaltered expression of genes that regulate programmed cell death orapoptosis (BCL2L1, PMIAP1 also known as NOXA), transporter proteins(SLC25A1 [citrate transporter], SLC25A13 [aspartate-glutamateexchanger], SLC25A19 [thiamine pyrophosphate transporter] and SLC25A22[glutamate-hydrogen cotransporter]) and mitochondrial matrix transportproteins (MFN1, TIMM44 and TOMM40). The activities of these transportersplay important role in the regulation of precursors essential for theKreb's cycle and maintenance of mitochondrial oxidative phosphorylation.These results indicate that exposure of diabetic HASMC cells to CoenzymeQ10 is associated with changes in expression of cytoplasmic andmitochondrial genes, which in turn is consistent with Coenzyme Q10providing a therapeutic benefit in the treatment of diabetes.

A comparison of the data obtained by treating HASMC cells and HK-2 cellswith Coenzyme Q10 or in a hyperglycemic environment reveals that 4 geneswere commonly regulated by Coenzyme Q10 in both cell lines (e.g.,PIK3C2B and SELL in the gene expression assay and TOMM40 and TSPO in themitochondrial array assay). These results demonstrate that treatment ofcells with Coenzyme Q10 in a diabetic environment is associated withaltered expression of genes that are known to be involved in the causeor treatment of diabetes.

Example 46 In Vivo Effects of Coenzyme Q10 Administration on PancreaticCancer

An intravenously administered formulation of coenzyme Q10 was evaluatedfor treating pancreatic cancer in an animal model. Rats with inducedpancreatic cancer were randomized into groups and received one of thefollowing 9 treatments:

-   -   Group A: control    -   Group B: saline solution    -   Group C: vehicle    -   Group D: 5 mg/kg coenzyme Q10    -   Group E: 10 mg/kg coenzyme Q10    -   Group F: 25 mg/kg coenzyme Q10    -   Group G: 50 mg/kg coenzyme Q10    -   Group H: 5 mg/kg Doxorubicin    -   Group I: 50 mg/kg coenzyme Q10 and 5 mg/kg Doxorubicin

After 28 days, all animals in Groups A and B and the majority of theanimals in Group C had died. In contrast, most of the animals in GroupsD, E and F remained alive, with those animals receiving the higher doseof coenzyme Q10 remaining alive longer. Indeed, all of the animalsreceiving the highest dose of coenzyme Q10 (Group G) remained alive at28 days. These data demonstrate an overall dose response curve in whichthose animals receiving higher doses had a higher survival rate.

To evaluate the effectiveness of coenzyme Q10 in treating pancreaticcancer in combination with Doxorubicin, Group H was administeredDoxorubicin alone, while Group I was administered the combination ofDoxorubicin and coenzyme Q10. After 28 days, a significant number of theanimals in Group H had died due to the toxicity of Doxorubicin, whilethose animals in Group I had an increased survival rate. These datasuggest that, in addition to increasing the survival rate associatedwith pancreatic cancer, coenzyme Q10 can also mitigate the toxic sideeffects of a chemotherapeutic regimen.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments and methods described herein. Such equivalents are intendedto be encompassed by the scope of the following claims.

1. A method for treating, alleviating symptoms of, inhibitingprogression of, or preventing an oncological disorder in a mammal, themethod comprising: administering to the mammal in need thereof atherapeutically effective amount of pharmaceutical compositioncomprising at least one environmental influencer (env-influencer),wherein the environmental influencer selectively elicits, in a cancerouscell of the mammal, a cellular metabolic energy shift from glycolysis tomitochondrial oxidative phosphorylation, towards levels observed in anormal cell of the mammal under normal physiological conditions.
 2. Themethod of claim 1, wherein the environmental influencer does notsubstantially elicit, in non-cancerous cells of the mammal, the cellularmetabolic energy shift from glycolysis to mitochondrial oxidativephosphorylation.
 3. The method of claim 1, wherein the mammal is human(or a non-human mammal).
 4. The method of claim 1, wherein theenvironmental influencer is not Coenzyme Q10, or its metabolites oranalogs thereof, including analogs having no or at least one isoprenylrepeats.
 5. The method of claim 1, wherein the oncological disorder isresponsive or sensitive to treatment by Coenzyme Q10 or its metabolitesor analogs thereof.
 6. The method of claim 1, wherein the environmentalinfluencer induces apoptosis or cell death mechanism in the cancerouscell.
 7. The method of claim 1, wherein the environmental influencerinhibits angiogenesis in the cancerous cell.
 8. The method of claim 1,wherein the environmental influencer induces a modulation of theimmune-related elements within the microenvironment in the cancerouscell.
 9. The method of claim 1, wherein the environmental influencerinduces a change in cell cycle control in the cancerous cell.
 10. Themethod of claim 1, wherein the environmental influencer comprises: (a)benzoquinone or at least one molecule that facilitates the biosynthesisof the benzoquinone ring, and (b) at least one molecule that facilitatesthe synthesis of and/or attachment of isoprenoid units to thebenzoquinone ring.
 11. The method of claim 10, wherein said at least onemolecule that facilitates the biosynthesis of the benzoquinone ringcomprises: L-Phenylalanine, DL-Phenylalanine, D-Phenylalanine,L-Tyrosine, DL-Tyrosine, D-Tyrosine, 4-hydroxy-phenylpyruvate,3-methoxy-4-hydroxymandelate (vanillylmandelate or VMA), vanillic acid,pyridoxine, or panthenol.
 12. The method of claim 10, wherein said atleast one molecule that facilitates the synthesis of and/or attachmentof isoprenoid units to the benzoquinone ring comprises: phenylacetate,4-hydroxy-benzoate, mevalonic acid, acetylglycine, acetyl-CoA, orfarnesyl.
 13. The method of claim 1, wherein the environmentalinfluencer comprises: (a) one or more of L-Phenylalanine, L-Tyrosine,and 4-hydroxyphenylpyruvate; and, (b) one or more of 4-hydroxy benzoate,phenylacetate, and benzoquinone.
 14. The method of claim 1, wherein theenvironmental influencer: (a) inhibits Bcl-2 expression and/or promotesCaspase-3 expression; and/or, (b) inhibits cell proliferation.
 15. Themethod of claim 1, wherein the environmental influencer is amultidimensional intracellular molecule (MIM).
 16. The method of claim15, wherein the MIM is selected from: alpha ketoglutarate/alphaketoglutaric acid, Malate/Malic acid, Succinate/Succinic acid,Glucosamine, Adenosine, Adenosine Diphosphate, Glucuronide/Glucuronicacid, Nicotinic Acid, Nicotinic Acid Dinucleotide,Alanine/Phenylalanine, Pyridoxine, Thiamine, or Flavin AdenineDinucleotide.
 17. The method of claim 1, wherein the environmentalinfluencer is an epimetabolic shifter.
 18. The method of claim 17,wherein the epimetabolic shifter is selected from: Transaldolase,Transketolase, Succinyl CoA synthase, Pyruvate Carboxylase, orRiboflavin.
 19. The method of claim 1, wherein the therapeuticallyeffective amount of said at least one environmental influencerdown-regulates anaerobic use of glucose and up-regulates mitochondrialoxidative phosphorylation.
 20. The method of claim 1, wherein the formof the environmental influencer administered to the human is differentthan the predominant form found in systemic circulation in the human.21. The method of claim 1, wherein the oncological disorder is selectedfrom the group consisting of: a leukemia, a lymphoma, a melanoma, acarcinoma and a sarcoma.
 22. The method of claim 1, further comprisingadministering to the mammal an additional therapeutic agent or treatmentregimen.
 23. A method for selectively blocking, in a cancerous cell of amammal in need of treatment for an oncological disorder, anaerobic useof glucose (glycolysis) and augmenting mitochondrial oxidativephosphorylation, the method comprising: administering to said mammal atherapeutically effective amount of at least one env-influencer toselectively block anaerobic use of glucose and to augment mitochondrialoxidative phosphorylation in said cancerous cell of the mammal, towardslevels observed in a normal cell of the mammal under normalphysiological conditions.
 24. The method of claim 23, furthercomprising: (1) up-regulating the expression of one or more genesselected from the group consisting of the genes set forth in Tables 2-4& 6-28 having a positive fold change; and/or, (2) down-regulating theexpression of one or more genes selected from the group consisting ofthe genes set forth in Tables 2-4 & 6-28 having a negative fold change.25. The method of claim 23, further comprising modulating the expressionof one or more genes selected from the group consisting of HNF4-alpha,Bcl-xl, Bcl-xS, BNIP-2, Bcl-2, Birc6, Bcl-2-L11, XIAP, BRAF, Bax, c-Jun,Bmf, PUMA, cMyc, transaldolase 1, COQ1, COQ3, COQ6, prenyltransferase,4-hydrobenzoate, neutrophil cytosolic factor 2, nitric oxide synthase2A, superoxide dismutase 2, VDAC, Bax channel, ANT, Cytochrome c,complex 1, complex II, complex III, complex IV, Foxo 3a, DJ-1, IDH-1,Cpt1C and Cam Kinase II.
 26. The method of any one of claims 23-25,wherein the oncological disorder is selected from the group consistingof a leukemia, a lymphoma, a melanoma, a carcinoma and a sarcoma. 27.The method of any one of claims 23-25, further comprising a treatmentregimen selected from the group consisting of surgery, radiation,hormone therapy, antibody therapy, therapy with growth factors,cytokines, and chemotherapy.
 28. A method for identifying an effectiveenvironmental influencer for treating, alleviating symptoms of,inhibiting progression of, or preventing an oncological disorder in amammal, the method comprising: (1) obtaining a diseased biologicalsample comprising cancer cells of the oncological disorder, and a normalbiological sample comprising no cancer cells; (2) contacting thediseased and normal biological samples with a candidate environmentalinfluencer; (3) determining the level of expression of one or moremarkers present in the diseased and normal biological samples, whereinthe marker is selected from the group consisting of the markers listedin Tables 2-4 & 6-28 having a positive fold change and/or having anegative fold change; (4) comparing the level of expression of the oneof more markers in the diseased and normal biological samples; whereinan effective environmental influencer is identified as the candidateenvironmental influencer that increases the level of expression of theone or more markers having a positive fold change and/or decreases thelevel of expression of the one or more markers having a negative foldchange, in the diseased biological sample but substantially not in thenormal biological sample.
 29. A method for treating, alleviatingsymptoms of, inhibiting progression of, or preventing an oncologicaldisorder in a mammal, the method comprising: (1) obtaining a diseasedbiological sample comprising cancer cells of the oncological disorder,and a normal biological sample comprising no cancer cells; (2)contacting the diseased and normal biological samples with a candidateenvironmental influencer; (3) determining the level of expression of oneor more markers present in the diseased and normal biological samples,wherein the marker is selected from the group consisting of the markerslisted in Tables 1-28 having a positive fold change and/or having anegative fold change; (4) comparing the level of expression of the oneof more markers in the diseased and normal biological samples; whereinan effective environmental influencer is identified as the candidateenvironmental influencer that increases the level of expression of theone or more markers having a positive fold change and/or decreases thelevel of expression of the one or more markers having a negative foldchange, in the diseased biological sample but substantially not in thenormal biological sample; (5) administering to the mammal the effectiveenvironmental influencer; thereby treating the oncological disorder inthe mammal.
 30. A method for identifying an effective environmentalinfluencer for treating, alleviating symptoms of, inhibiting progressionof, or preventing an oncological disorder in a mammal, the methodcomprising: (1) obtaining a diseased biological sample comprising cancercells of the oncological disorder, and a normal biological samplecomprising no cancer cells; (2) contacting the diseased and normalbiological samples with a candidate environmental influencer; (3)determining the level of glycolysis and mitochondrial oxidativephosphorylation in the diseased and normal biological samples, beforeand after contacting the candidate environmental influencer; wherein aneffective environmental influencer is identified as the candidateenvironmental influencer that increases the level of mitochondrialoxidative phosphorylation and/or decreases the level of glycolysis, inthe diseased biological sample but substantially not in the normalbiological sample.
 31. A method for treating, alleviating symptoms of,inhibiting progression of, or preventing an oncological disorder in amammal, the method comprising: (1) obtaining a diseased biologicalsample comprising cancer cells of the oncological disorder, and a normalbiological sample comprising no cancer cells; (2) contacting thediseased and normal biological samples with a candidate environmentalinfluencer; (3) determining the level of glycolysis and mitochondrialoxidative phosphorylation in the diseased and normal biological samples,before and after contacting the candidate environmental influencer,wherein an effective environmental influencer is identified as thecandidate environmental influencer that increases the level ofmitochondrial oxidative phosphorylation and/or decreases the level ofglycolysis, in the diseased biological sample but substantially not inthe normal biological sample; and, (4) administering to the mammal theeffective environmental influencer; thereby treating the oncologicaldisorder in the mammal.
 32. The method of claim 30 or 31, wherein thelevel of glycolysis is measured as ECAR, and/or wherein the level ofmitochondrial oxidative phosphorylation is measured as OCR.
 33. Themethod of claim 23, wherein the env-influencer is not coenzyme Q10. 34.A method for identifying a Multidimensional Intracellular Molecule,comprising: (1) contacting a cell with an endogenous molecule; (2)monitoring the effect of the endogenous molecule on a cellularmicroenvironment profile; and (3) identifying an endogenous moleculethat induces a change to the cellular microenvironment profile; therebyidentifying a Multidimensional Intracellular Molecule.
 35. The method ofclaim 34, further comprising: (1) comparing the effects of theendogenous molecule on the cellular microenvironment profile of adiseased cell and a normal control cell; and (2) identifying anendogenous molecule that differentially induces a change to the cellularmicroenvironment profile of the diseased cell as compared to the normalcontrol cell.
 36. The method of claim 34 or 35, wherein the effect onthe cellular microenvironment profile is monitored by measuring a changein the level or activity of a cellular molecule selected from the groupconsisting of mRNA, protein, lipid and metabolite.
 37. A method ofidentifying an Epimetabolic shifter, comprising: (1) comparing molecularprofiles for two or more cells or tissues, wherein the two or more cellsor tissues display differential disease states; (2) identifying amolecule from the molecular profiles for which a change in levelcorrelates to the disease state; (3) introducing the molecule to a cell;and (4) evaluating the ability of the molecule to shift the metabolicstate of a cell; wherein a molecule capable of shifting the metabolicstate of a cell is identified as an Epimetabolic shifter.
 38. The methodof claim 37, wherein the molecular profile is selected from the groupconsisting of a metabolite profile, lipid profile, protein profile orRNA profile.
 39. The method of claim 38, wherein the molecule does notnegatively effect the health or growth of a normal cell.
 40. A method ofidentifying an agent that is effective in treating an oncologicaldisorder, comprising: (1) providing a candidate environmentalinfluencer; (2) determining the ability of the candidate environmentalinfluencer to shift the metabolic state of a cell; and (3) determiningwhether the candidate environmental influencer is effective in treatingthe oncological disorder; wherein the candidate environmental influencercapable of shifting the metabolic state of the cell and is effective intreating the oncological disorder is identified as the agent effectivein treating the oncological disorder.
 41. The method of claim 40,wherein shift in the metabolic state of the cell is determined bymeasuring a change in one or more of mRNA expression, proteinexpression, lipid levels, metabolite levels, levels of bioenergeticmolecules, cellular energetics, mitochondrial function and mitochondrialnumber.
 42. A composition comprising an agent identified according tothe method of any one of claims 34-41.
 43. A method for treating,alleviating symptoms of, inhibiting progression of, or preventing aCoQ10 responsive disorder in a mammal, the method comprising:administering to the mammal in need thereof a therapeutically effectiveamount of pharmaceutical composition comprising at least oneenvironmental influencer (env-influencer), wherein the environmentalinfluencer selectively elicits, in a disease cell of the mammal, acellular metabolic energy shift towards levels of glycolysis andmitochondrial oxidative phosphorylation observed in a normal cell of themammal under normal physiological conditions.
 44. The method of claim43, wherein the CoQ10 responsive disorder is an oncological disorder.